Materials and methods for treatment of usher syndrome type 2a

ABSTRACT

The present application provides materials and methods for treating a patient with Usher Syndrome Type 2A, both ex vivo and in vivo; materials and methods for editing a USH2A gene in a human cell; materials and methods for editing an USH2A gene containing an IVS40 mutation; materials and methods for treating a patient with an USH2A gene containing an IVS40 mutation; and a method for deleting a sequence comprising an IVS40 mutation within a USH2A gene of a cell. The present application also provides one or more gRNAs or sgRNAs for editing an USH2A gene containing an IVS40 mutation. The present application provides a therapeutic for treating a patient with Usher Syndrome Type 2A. The present application also provides a kit for treating a patient with Usher Syndrome Type 2A.

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/IB2018/060546, filed Dec. 21, 2018, which claims the benefit of U.S.Provisional Application No. 62/609,333 filed Dec. 21, 2017; and U.S.Provisional Application No. 62/746,226 filed Oct. 16, 2018. The entirecontents of these applications are incorporated herein by reference intheir entirety.

FIELD

The present application provides materials and methods for treatingUsher Syndrome Type 2A.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format via EFS-Web, and is herebyincorporated by reference in its entirety. Said ASCII copy, created onJun. 18, 2020, is named Sequence_Listing_CBTN_003PCCN.txt and is11399039 bytes in size.

BACKGROUND

Usher syndrome is a condition that affects both hearing and vision. Themajor symptoms of Usher syndrome are hearing loss and an eye disordercalled retinitis pigmentosa, which causes night-blindness and a loss ofperipheral vision through the progressive degeneration of the retina.Many people with Usher syndrome also have severe balance problems.

There are currently no adequate treatments for Usher Syndrome that canefficiently halt or slow the progression of the visual loss associatedwith the disease and there remains a critical need for developing safeand effective treatments for Usher Syndrome.

SUMMARY

The present disclosure presents a novel method to ameliorate, if noteliminate, Usher Syndrome Type 2A. The novel approach targets a mutationin the USH2A gene, such as an IVS40 mutation, with a method resulting inthe disruption of a sequence used as a splice donor site encoded by agene containing the mutation. The splice donor site causes incorrectsplicing. Furthermore, in some cases, the treatment can be effected witha small number of treatments and, in some cases, with a singletreatment. The resulting therapy can ameliorate Usher Syndrome Type 2Aassociated with an IVS40 mutation, or in some cases, can eliminate UsherSyndrome Type 2A associated with an IVS40 mutation.

Provided herein is a method for editing an USH2A gene in a human cell,the method comprises: introducing into the human cell one or moredeoxyribonucleic acid (DNA) endonuclease, thereby effecting one or moresingle-strand breaks (SSBs) or double-strand breaks (DSBs) within ornear the USH2A gene or a DNA sequence encoding a regulatory sequence ofthe USH2A gene that results in a correction thereby creating an editedhuman cell.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: introducing into the human cellone or more DNA endonuclease, thereby effecting one or more SSBs or DSBswithin or near intron 40 of the USH2A gene that results in a correctionthereby creating an edited human cell.

Also provided herein is a method for editing an USH2A gene in a humancell, the method comprises: introducing into the human cell one or moreDNA endonuclease, thereby effecting one or more SSBs or DSBs within ornear the USH2A gene or a DNA sequence encoding regulatory sequence ofthe USH2A gene that results in a modulation of expression or function ofthe USH2A gene thereby creating an edited human cell.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: introducing into the human cellone or more DNA endonuclease, thereby effecting one or more SSBs or DSBswithin or near intron 40 of the USH2A gene that results in a modulationof expression or function of the USH2A gene thereby creating an editedhuman cell.

Also provided herein is an in vivo method for treating a patient withUsher Syndrome Type 2A. The method comprises: editing an USH2A genecontaining an IVS40 mutation in a cell of the patient.

Also provided herein are one or more guide ribonucleic acids (gRNAs) forediting an USH2A gene containing an IVS40 mutation in a cell from apatient with Usher Syndrome Type 2A. The one or more gRNAs comprise aspacer sequence selected from the group consisting of nucleic acidsequences in SEQ ID NOs: 5272-5319, 5321, 5323, 5325, 5327-5328, 5443and 5446-5461 of the Sequence Listing.

Also provided herein is a therapeutic for treating a patient with UsherSyndrome Type 2A, the therapeutic comprising at least one or more gRNAsfor editing an USH2A gene containing an IVS40 mutation. The one or moregRNAs comprise a spacer sequence selected from the group consisting ofnucleic acid sequences in SEQ ID NOs: 5272-5319, 5321, 5323, 5325,5327-5328, 5443 and 5446-5461 of the Sequence Listing.

Also provided herein is a therapeutic for treating a patient with UsherSyndrome Type 2A, the therapeutic formed by a method comprising:introducing one or more DNA endonucleases; introducing one or more gRNAor one or more single-molecule guide RNA (sgRNA) for editing an USH2Agene containing an IVS40 mutation; and optionally introducing one ormore donor template. The one or more gRNAs or sgRNAs comprise a spacersequence selected from the group consisting of nucleic acid sequences inSEQ ID NOs: 5272-5319, 5321, 5323, 5325, 5327-5328, 5443 and 5446-5461of the Sequence Listing.

Also provided herein is a kit for treating a patient with Usher SyndromeType 2A in vivo. The kit comprises one or more gRNAs or sgRNAs forediting an USH2A gene containing an IVS40 mutation, one or more DNAendonucleases; and optionally, one or more donor template. The one ormore gRNAs or sgRNAs comprise a spacer sequence selected from the groupconsisting of nucleic acid sequences in SEQ ID NOs: 5272-5319, 5321,5323, 5325, 5327-5328, 5443 and 5446-5461 of the Sequence Listing.

Also provided herein is a gRNA or sgRNA comprising SEQ ID NO: 5321.

Also provided herein is a gRNA or sgRNA comprising SEQ ID NO: 5323.

Also provided herein is a gRNA or sgRNA comprising SEQ ID NO: 5325.

Also provided herein is a gRNA or sgRNA comprising SEQ ID NO: 5327.

Also provided herein is a gRNA or sgRNA comprising SEQ ID NO: 5328.

Also provided herein is a gRNA or sgRNA comprising SEQ ID NO: 5321 andany one of SEQ ID NOs: 5267-5269.

Also provided herein is a gRNA or sgRNA comprising SEQ ID NO: 5323 andany one of SEQ ID NOs: 5267-5269.

Also provided herein is a gRNA or sgRNA comprising SEQ ID NO: 5325 andany one of SEQ ID NOs: 5267-5269.

Also provided herein is a gRNA or sgRNA comprising SEQ ID NO: 5327 andany one of SEQ ID NOs: 5267-5269.

Also provided herein is a gRNA or sgRNA comprising SEQ ID NO: 5328 andany one of SEQ ID NOs: 5267-5269.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: editing the USH2A gene containingthe IVS40 mutation using a gRNA or sgRNA comprising SEQ ID NO: 5321.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: editing the USH2A gene containingthe IVS40 mutation using a gRNA or sgRNA comprising SEQ ID NO: 5323.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: editing the USH2A gene containingthe IVS40 mutation using a gRNA or sgRNA comprising SEQ ID NO: 5325.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: editing the USH2A gene containingthe IVS40 mutation using a gRNA or sgRNA comprising SEQ ID NO: 5327.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: editing the USH2A gene containingthe IVS40 mutation using a gRNA or sgRNA comprising SEQ ID NO: 5328.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: editing the USH2A gene containingthe IVS40 mutation using a gRNA or sgRNA comprising SEQ ID NO: 5321 andany one of SEQ ID NOs: 5267-5269.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: editing the USH2A gene containingthe IVS40 mutation using a gRNA or sgRNA comprising SEQ ID NO: 5323 andany one of SEQ ID NOs: 5267-5269.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: editing the USH2A gene containingthe IVS40 mutation using a gRNA or sgRNA comprising SEQ ID NO: 5325 andany one of SEQ ID NOs: 5267-5269.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: editing the USH2A gene containingthe IVS40 mutation using a gRNA or sgRNA comprising SEQ ID NO: 5327 andany one of SEQ ID NOs: 5267-5269.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: editing the USH2A gene containingthe IVS40 mutation using a gRNA or sgRNA comprising SEQ ID NO: 5328 andany one of SEQ ID NOs: 5267-5269.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering agRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises SEQ IDNO: 5321.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering agRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprise SEQ IDNO: 5323.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering agRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprise SEQ IDNO: 5325.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering agRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprise SEQ IDNO: 5327.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering agRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprise SEQ IDNO: 5328.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering agRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprise SEQ IDNO: 5321 and any one of SEQ ID NOs: 5267-5269.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering agRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprise SEQ IDNO: 5323 and any one of SEQ ID NOs: 5267-5269.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering agRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprise SEQ IDNO: 5325 and any one of SEQ ID NOs: 5267-5269.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering agRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprise SEQ IDNO: 5327 and any one of SEQ ID NOs: 5267-5269.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering agRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprise SEQ IDNO: 5328 and any one of SEQ ID NOs: 5267-5269.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: deleting a sequence comprising theIVS40 mutation using a first gRNA or sgRNA comprising SEQ ID NO: 5295and a second gRNA or sgRNA comprising SEQ ID NO: 5279.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: deleting a sequence comprising theIVS40 mutation using a first gRNA or sgRNA comprising SEQ ID NO: 5294and a second gRNA or sgRNA comprising SEQ ID NO: 5300.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: deleting a sequence comprising theIVS40 mutation using a first gRNA or sgRNA comprising SEQ ID NO: 5295and a second gRNA or sgRNA comprising SEQ ID NO: 5300.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: deleting a sequence comprising theIVS40 mutation using a first gRNA or sgRNA comprising SEQ ID NO: 5290and a second gRNA or sgRNA comprising SEQ ID NO: 5300.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: deleting a sequence comprising theIVS40 mutation using a first gRNA or sgRNA comprising SEQ ID NO: 5277and a second gRNA or sgRNA comprising SEQ ID NO: 5300.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering afirst gRNA or sgRNA and second gRNA or sgRNA to the patient, wherein thefirst gRNA or sgRNA comprise SEQ ID NO: 5295 and the second gRNA orsgRNA comprise SEQ ID NO: 5279.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering afirst gRNA or sgRNA and second gRNA or sgRNA to the patient, wherein thefirst gRNA or sgRNA comprise SEQ ID NO: 5294 and the second gRNA orsgRNA comprise SEQ ID NO: 5300.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering afirst gRNA or sgRNA and second gRNA or sgRNA to the patient, wherein thefirst gRNA or sgRNA comprise SEQ ID NO: 5295 and the second gRNA orsgRNA comprise SEQ ID NO: 5300.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering afirst gRNA or sgRNA and second gRNA or sgRNA to the patient, wherein thefirst gRNA or sgRNA comprise SEQ ID NO: 5290 and the second gRNA orsgRNA comprise SEQ ID NO: 5300.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering afirst gRNA or sgRNA and second gRNA or sgRNA to the patient, wherein thefirst gRNA or sgRNA comprise SEQ ID NO: 5277 and the second gRNA orsgRNA comprise SEQ ID NO: 5300.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: deleting a sequence comprising theIVS40 mutation using a first gRNA or sgRNA comprising SEQ ID NO: 5452and a second gRNA or sgRNA comprising SEQ ID NO: 5449.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: deleting a sequence comprising theIVS40 mutation using a first gRNA or sgRNA comprising SEQ ID NO: 5453and a second gRNA or sgRNA comprising SEQ ID NO: 5449.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: deleting a sequence comprising theIVS40 mutation using a first gRNA or sgRNA comprising SEQ ID NO: 5455and a second gRNA or sgRNA comprising SEQ ID NO: 5457.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: deleting a sequence comprising theIVS40 mutation using a first gRNA or sgRNA comprising SEQ ID NO: 5452and a second gRNA or sgRNA comprising SEQ ID NO: 5451.

Also provided herein is a method for editing an USH2A gene containing anIVS40 mutation. The method comprises: deleting a sequence comprising theIVS40 mutation using a first gRNA or sgRNA comprising SEQ ID NO: 5448and a second gRNA or sgRNA comprising SEQ ID NO: 5449.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering afirst gRNA or sgRNA and second gRNA or sgRNA to the patient, wherein thefirst gRNA or sgRNA comprise SEQ ID NO: 5452 and the second gRNA orsgRNA comprise SEQ ID NO: 5449.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering afirst gRNA or sgRNA and second gRNA or sgRNA to the patient, wherein thefirst gRNA or sgRNA comprise SEQ ID NO: 5453 and the second gRNA orsgRNA comprise SEQ ID NO: 5449.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering afirst gRNA or sgRNA and second gRNA or sgRNA to the patient, wherein thefirst gRNA or sgRNA comprise SEQ ID NO: 5455 and the second gRNA orsgRNA comprise SEQ ID NO: 5457.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation. The method comprises: administering afirst gRNA or sgRNA and second gRNA or sgRNA to the patient, wherein thefirst gRNA or sgRNA comprise SEQ ID NO: 5452 and the second gRNA orsgRNA comprise SEQ ID NO: 5451.

Also provided herein is a method for treating a patient with an USH2Agene containing an IVS40 mutation The method comprises: administering afirst gRNA or sgRNA and second gRNA or sgRNA to the patient, wherein thefirst gRNA or sgRNA comprise SEQ ID NO: 5448 and the second gRNA orsgRNA comprise SEQ ID NO: 5449.

It is understood that the inventions described in this specification arenot limited to the examples summarized in this Summary. Various otheraspects are described and exemplified herein. This Summary is notintended to limit the scope of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of materials and methods for treatment of Usher Syndromedisclosed and described in this specification can be better understoodby reference to the accompanying figures, in which:

FIGS. 1A-B depict the type II CRISPR/Cas system.

FIG. 1A depicts the type II CRISPR/Cas system including gRNA.

FIG. 1B depicts the type II CRISPR/Cas system including sgRNA.

FIGS. 2A-F show the single guide RNA (sgRNA) sequence, the target DNAsequence, and the reverse strand of the target DNA sequence to which thesgRNA binds, for each of 57 sgRNA sequences.

FIGS. 2A-B show the single guide RNA (sgRNA) sequence, for each of 57sgRNA sequences.

FIGS. 2C-D show the target DNA sequence, for each of 57 sgRNA sequences.

FIGS. 2E-F show the reverse strand of the target DNA sequence to whichthe sgRNA binds, for each of 57 sgRNA sequences.

FIGS. 2G-I show the single guide RNA (sgRNA) sequence, the target DNAsequence, and the reverse strand of the target DNA sequence to which thesgRNA binds, for a sgRNA sequence.

FIG. 2G shows the single guide RNA (sgRNA) sequence for a sgRNAsequence.

FIG. 2H shows the target DNA sequence for a sgRNA sequence.

FIG. 2I shows the reverse strand of the target DNA sequence to which thesgRNA binds, for a sgRNA sequence.

FIGS. 2J-L show the single guide RNA (sgRNA) sequence, the target DNAsequence, and the reverse strand of the target DNA sequence to which thesgRNA binds, for each of 16 sgRNA sequences.

FIG. 2J shows the single guide RNA (sgRNA) sequence, for each of 16sgRNA sequences.

FIG. 2K shows the target DNA sequence, for each of 16 sgRNA sequences.

FIG. 2L shows the reverse strand of the target DNA sequence to which thesgRNA binds, for each of 16 sgRNA sequences.

FIG. 3 shows a diagram depicting the IVS40 mutation located in intron 40of the USH2A gene, the result of editing the IVS40 mutation in intron 40of the USH2A gene, and the result of not editing the IVS40 mutation inintron 40 of the USH2A gene.

FIGS. 4A-B show an IVS40 mutation introduced into genomic DNA viahomology directed repair (HDR); the IVS40 mutation is a singlenucleotide mutation (A to G) in intron 40 of the human USH2A gene.

FIG. 4A shows an IVS40 mutation introduced into genomic DNA via HDR.

FIG. 4B shows the IVS40 mutation as a single nucleotide mutation (A toG) in intron 40 of the human USH2A gene.

FIGS. 5A-B depict the binding regions of several USH2A IVS40 mutationtargeting sgRNAs and the on-target and off-target editing efficienciesfor each of the USH2A IVS40 mutation targeting sgRNAs.

FIG. 5A depicts the binding regions of several sgRNA spacer regions (SEQID NOs: 5321, 5323, 5325, 5327, and 5328) around the IVS40 mutation ofthe USH2A gene. The pointed end is the region adjacent to the PAMsequence in the genomic DNA.

FIG. 5B shows on-target and off-target editing efficiencies for each ofthe USH2A IVS40 mutation targeting sgRNAs depicted in FIG. 5A.

FIGS. 6A-C show the location relative to the IVS40 mutation, theposition relative to the IVS40 nucleotide, and the on-target editingefficiency for sgRNAs that (1) associate with SpCas9 or SaCas9 andeither (2) overlap with the IVS40 mutation, bind upstream of the IVS40mutation, or bind downstream of the IVS40 mutation. For the positionrelative to the IVS40 nucleotide, −8-(+11) indicates that the sgRNAbinds between position 8 nucleotides upstream of the IVS40 mutation to11 nucleotides downstream of the IVS40. For the position relative to theIVS40 nucleotide, −571-590 indicates that the sgRNA binds betweenposition 571 nucleotides upstream of the IVS40 mutation to 590nucleotides upstream of the IVS40. For the position relative to theIVS40 nucleotide, +744-763 indicates that the sgRNA binds betweenposition 744 nucleotides downstream of the IVS40 mutation to 763nucleotides downstream of the IVS40.

FIGS. 6A-B show the location relative to the IVS40 mutation, theposition relative to the IVS40 nucleotide, and the on-target editingefficiency for each of 52 sgRNAs that associate with SpCas9 and either(1) overlap with the IVS40 mutation, (2) bind upstream of the IVS40mutation, or (3) bind downstream of the IVS40 mutation.

FIG. 6C shows the location relative to the IVS40 mutation, the positionrelative to the IVS40 nucleotide, and the on-target editing efficiencyfor each of 16 sgRNAs that associate with SaCas9 and either (1) bindupstream of the IVS40 mutation, or (2) bind downstream of the IVS40mutation.

FIGS. 7A-F show the binding of a first sgRNA upstream of the IVS40mutation and the binding of a second sgRNA downstream of the IVS40mutation and 5 possible editing outcomes from using dual sgRNAs.

FIG. 7A depicts the binding of a first sgRNA upstream of the IVS40mutation and the binding of a second sgRNA downstream of the IVS40mutation.

FIG. 7B depicts unedited genomic DNA comprising the IVS40 mutation.

FIG. 7C depicts the editing of genomic DNA by either (1) a first sgRNAthat binds upstream of the IVS40 mutation; or (2) a second sgRNA thatbinds downstream of the IVS40 mutation.

FIG. 7D depicts the editing of genomic DNA by both (1) a first sgRNAthat binds upstream of the IVS40 mutation; and (2) a second sgRNA thatbinds downstream of the IVS40 mutation, but editing does not result in adeletion.

FIG. 7E depicts the editing of genomic DNA by both (1) a first sgRNAthat binds upstream of the IVS40 mutation; and (2) a second sgRNA thatbinds downstream of the IVS40 mutation and the editing results in adeletion.

FIG. 7F depicts the editing of genomic DNA by both (1) a first sgRNAthat binds upstream of the IVS40 mutation; and (2) a second sgRNA thatbinds downstream of the IVS40 mutation, but editing does not result in adeletion. Instead, editing results in an inversion.

FIG. 8 is a scheme for performing quantitative analysis of deletionsusing ddPCR.

FIGS. 9A-B show the deletion frequency and resulting deletion size forselected different dual sgRNA. The sgRNAs included in FIGS. 9A-B thatmake up each dual sgRNA associate with SpCas9.

FIG. 9A is a table showing the deletion frequency and resulting deletionsize for selected different dual sgRNA.

FIG. 9B is a graph showing the deletion frequency and resulting deletionsize for selected different dual sgRNA.

FIG. 10 depicts the pAAV-U6 plasmid, which can be engineered to encodesgRNAs that associate with SaCas9.

FIGS. 11A-B show the deletion frequency and resulting deletion size forselected different dual sgRNA. The sgRNAs included in FIGS. 11A-B thatmake up each dual sgRNA associate with SaCas9.

FIG. 11A is a table showing the deletion frequency and resultingdeletion size for selected different dual sgRNA.

FIG. 11B is a graph showing the deletion frequency and resultingdeletion size for selected different dual sgRNA.

FIGS. 12A-C show the splicing reporter plasmid, pET01; a schematicrepresentation of two different USH2A DNA inserts; and a gel image ofRT-PCR products amplified from mRNA of HEK 293 SpCas9 positive cellstransfected with 1 of 2 different splice reporter plasmids and 1 of 4different USH2A IVS40 mutation targeting sgRNAs.

FIG. 12A depicts the splicing reporter plasmid, pET01.

FIG. 12B depicts two different USH2A DNA inserts (part of mutant intron40 or part of wild-type intron 40) and vector elements around theinserts. The corresponding RNA splice products (mutant splice product orwild-type splice product) are also depicted.

FIG. 12C shows a gel image of RT-PCR products amplified from mRNA of HEK293 SpCas9 positive cells transfected with 1 of 2 different splicereporter plasmids (a plasmid comprising part of mutant intron 40 or aplasmid comprising part of wild-type intron 40) and 1 of 4 differentUSH2A IVS40 mutation-targeting sgRNAs (sgRNAs comprising 5321, 5323,5325, or 5327).

FIGS. 13A-C show constructs used for a blue fluorescent protein (BFP)splicing reporter assay and possible effects that genome editing canhave on the constructs and BFP gene expression therefrom.

FIG. 13A depicts a construct that expresses BFP using a phosphoglyceratekinase promoter. The BFP gene comprises part of the wild-type sequenceof the USH2A gene intron 40 upstream of the BFP ORF. BFP expression isexpected.

FIG. 13B depicts a construct that expresses BFP using a phosphoglyceratekinase promoter. The BFP gene comprises part of the IVS40 mutantsequence of the USH2A gene intron 40 upstream of the BFP ORF. BFPexpression is not expected.

FIG. 13C depicts the construct from FIG. 13B in a first configuration,before genome editing, and in a second configuration, after genomeediting. After genome editing, BFP expression is expected.

FIG. 14 shows the percent of live cells that expressed BFP after beingsubjected to a BFP splicing reporter assay. Data for editing strategiesthat use single sgRNAs and dual sgRNAs are shown. sgRNAs were pairedwith SpCas9.

FIG. 15 shows the percent of live cells that expressed BFP after beingsubjected to a BFP splicing reporter assay. Data for editing strategiesthat use dual sgRNAs are shown. These pairs of sgRNAs were paired withSaCas9.

FIG. 16 shows the percent of GFP positive cells that expressed BFP afterbeing subjected to a BFP splicing reporter assay. Data for editingstrategies that use single sgRNAs and dual sgRNAs are shown. ThesesgRNAs were paired with either SpCas9 or SaCas9. Negative controls forediting are also shown.

FIGS. 17A-B show the sites bound by primers and probes used in ddPCRassays of USH2A transcripts.

FIG. 17A shows a sequence of mRNA transcribed from an IVS40 mutant USH2Agene. Also shown are locations where primers and probes bind to thecorresponding cDNA.

FIG. 17B shows a sequence of mRNA transcribed from a wild-type USH2Agene (or an edited USH2A gene). Also shown are locations where primersand probes bind to the corresponding cDNA.

FIG. 18 shows the percent of corrected and uncorrected transcripts inIVS40 mutant cells that have been subjected to genome editing accordingto the present disclosure. Data for editing strategies that use singlesgRNAs and dual sgRNAs are shown. These sgRNAs were paired with SpCas9.Negative controls are also shown.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NOs: 1-612 are Cas endonuclease ortholog sequences.

SEQ ID NOs: 613-4696 are microRNA sequences.

SEQ ID NOs: 4697-5265 are AAV serotype sequences.

SEQ ID NO: 5266 is a humanUSH2A nucleotide sequence.

SEQ ID NOs: 5267-5269 show sample sgRNA backbone sequences that SpCas9is complexed with.

SEQ ID NO: 5270 is a sample guide RNA (gRNA) for a Streptococcuspyogenes Cas9 endonuclease.

SEQ ID NO: 5271 shows a known family of homing endonuclease, asclassified by its structure.

SEQ ID NOs: 5272-5319 are 20 bp spacer sequences for targeting regionsupstream and downstream of the IVS40 mutation, within or near intron 40of the USH2A gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NO: 5320 is a 20 bp spacer sequence for targeting within or neara USH2A gene or other DNA sequence that encodes a regulatory sequence ofthe USH2A gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NO: 5321 is a 20 bp spacer sequence for targeting regionsupstream and downstream of the IVS40 mutation, within or near intron 40of the USH2A gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NO: 5322 is a 20 bp spacer sequence for targeting within or neara USH2A gene or other DNA sequence that encodes a regulatory sequence ofthe USH2A gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NO: 5323 is a 20 bp spacer sequence for targeting regionsupstream and downstream of the IVS40 mutation, within or near intron 40of the USH2A gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NO: 5324 is a 20 bp spacer sequence for targeting within or neara USH2A gene or other DNA sequence that encodes a regulatory sequence ofthe USH2A gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NO: 5325 is a 20 bp spacer sequence for targeting regionsupstream and downstream of the IVS40 mutation, within or near intron 40of the USH2A gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NO: 5326 is a 20 bp spacer sequence for targeting within or neara USH2A gene or other DNA sequence that encodes a regulatory sequence ofthe USH2A gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 5327-5328 are 20 bp spacer sequences for targeting regionsupstream and downstream of the IVS40 mutation, within or near intron 40of the USH2A gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 5329-5385 are sequences that represent the target DNAsequences, for each of 57 sgRNA sequences.

SEQ ID NOs: 5386-5442 are sequences that represent the reverse strandsof the target DNA sequence to which the sgRNA will bind, for each of 57sgRNA sequences.

SEQ ID NO: 5443 is an 18 bp spacer sequence for targeting regionsdownstream of the IVS40 mutation with a S. pyogenes Cas9 endonuclease.

SEQ ID NO: 5444 is a sequence that represents the target DNA sequencefor an 18 bp sgRNA sequence.

SEQ ID NO: 5445 is a sequence that represents the reverse strand of thetarget DNA sequence to which the sgRNA will bind for an 18 bp sgRNAsequence.

SEQ ID NOs: 5446-5461 are 20 bp spacer sequences for targeting regionsupstream and downstream of the IVS40 mutation, within or near intron 40of the USH2A gene with a Staphylococcus aureus Cas9 endonuclease.

SEQ ID NOs: 5462-5477 are sequences that represent the target DNAsequences, for each of 16 sgRNA sequences.

SEQ ID NOs: 5478-5493 are sequences that represent the reverse strandsof the target DNA sequence to which the sgRNA will bind, for each of 16sgRNA sequences.

SEQ ID NO: 5494 is a single-stranded HDR donor sequence.

SEQ ID NOs: 5495-5506 are PCR primer sequences.

SEQ ID NOs: 5507-5523 are sgRNA expressing plasmid sequences.

SEQ ID NO: 5524 is a sequence for pET01 comprising part of wild-typeintron 40 of USH2A.

SEQ ID NO: 5525 is a sequence for pET01 comprising part of mutant intron40 of USH2A.

SEQ ID NOs: 5526-5537 show sample sgRNA backbone sequences that SaCas9is complexed with.

SEQ ID NOs: 5538-5549 are sgRNA expressing plasmid sequences. The sgRNAsassociate with SpCas9.

SEQ ID NOs: 5550-5557 are sgRNA expressing plasmid sequences. The sgRNAsassociate with SaCas9.

SEQ ID NOs: 5558 and 5559 are an oligonucleotide sequences used toamplify a section of cDNA resulting from USH2A transcripts.

SEQ ID NOs: 5560 and 5561 are oligonucleotide probes used to detectcorrected or uncorrected USH2A cDNA sequences.

DETAILED DESCRIPTION

Applicants have discovered a novel method for treating Usher SyndromeType 2A, e.g., an Usher Syndrome Type 2A associated with an IVS40mutation in a USH2A gene. The method can result in slowing or reversingthe development of Usher Syndrome Type 2A or preventing development ofdisease in an individual.

Therapeutic Approach

The methods provided herein, regardless of whether a cellular, ex vivoor in vivo method can involve one or a combination of the followingmethods. One method involves disrupting the consensus sequence used as asplice donor site within or near the IVS40 mutation in the USH2A gene byinsertions and/or deletions that arise due to the non-homologous endjoining (NHEJ) pathway. In another method, the IVS40 mutation located inintron 40 of the USH2A gene is excised. In a third method, a mutantallele (e.g., an IVS40 mutation) is corrected by HDR.

The NHEJ strategy can involve inducing one single-stranded break ordouble-stranded break within or near the IVS40 mutation in the USH2Agene with one or more CRISPR endonucleases and a gRNA (e.g.,cRNA+tracrRNA, or sgRNA). This approach edits the sequence within ornear the IVS40 mutation and can disrupt the sequence that is causing theincorrect splicing. This method utilizes gRNAs or sgRNAs specific forthe IVS40 mutation in the USH2A gene.

The excision strategy can include a set of guide RNAs or sgRNAs thatbind upstream and downstream of the IVS40 mutation, within intron 40 ofthe USH2A gene and excise an area of the genome containing the IVS40mutation. This strategy can be expected to affect both the mutant (Mut)and the wild-type (WT) alleles, which is permissible with intronicmutations. The excision strategy can result in a shorter version of thenascent precursor messenger RNA (pre-mRNA, missing the dominant splicedonor creating mutation), which can be spliced correctly, leading to WTmRNA and expression of WT usherin protein in edited cells, as depictedin FIG. 3. The edited cell's function and survival can be expected toimprove in cases where enough supporting retinal structure is stillavailable. This method utilizes gRNAs or sgRNAs specific for the regionsupstream and downstream of the IVS40 mutation, within intron 40 of theUSH2A gene.

The deletions created by the excision strategy can be from 50 to 5000base pairs (bp) in size. For example, deletions can range from 50-100;50-250; 50-500; 50-1000; 50-1500; 50-2000; 200-500; 200-750; 200-1000;200-1100; 500-1,000; 1,000-1,500; 1,500-2,000; 1,000-2,000; 2,000-2,500;2,500-3,000; 3,000-3,500; 3,500-4,000; 4,000-4,500; 4,500-5,000 or50-2,900 base pairs in size.

The HDR strategy can involve inducing one or more single-stranded breaksor double-stranded breaks upstream and downstream of the IVS40 mutation,within or near intron 40 of the USH2A gene with one or more CRISPRendonucleases and a gRNA (e.g., crRNA+tracrRNA, or sgRNA), or two ormore single-stranded breaks or double-stranded breaks upstream anddownstream of the IVS40 mutation within or near intron 40 of the USH2Agene with one or more CRISPR endonucleases (Cas9, Cpf1 and the like) andtwo or more gRNAs, in the presence of a donor DNA template introducedexogenously to direct the cellular DSB response to Homology-DirectedRepair. The donor DNA template can be a short single-strandedoligonucleotide, a short double-stranded oligonucleotide, a long singleor double-stranded DNA molecule. The methods can provide gRNA pairs thatmake a deletion by cutting the gene twice, one gRNA cutting at the 5′end of the IVS40 mutation and the other gRNA cutting at the 3′ end ofthe IVS40 mutation that facilitates insertion of a new sequence from apolynucleotide donor template to replace the IVS40 mutation in the USH2Agene. The cutting can be accomplished by a pair of DNA endonucleasesthat each makes a DSB (one DSB on each end of the IVS40 mutation withinor near intron 40), or by multiple nickases that together make a DSB(one DSB on each end of the IVS40 mutation within or near intron 40).This method utilizes gRNAs or sgRNAs specific for regions upstream anddownstream of the IVS40 mutation, within intron 40 of the USH2A gene.This method also utilizes donor DNA molecules.

The advantages for the above strategies (disruption of RNA splicingconsensus sequence, excision, and HDR strategies) are similar, includingin principle both short and long term beneficial clinical and laboratoryeffects.

Such methods use endonucleases, such as CRISPR-associated (Cas9, Cpf1and the like) nucleases, to stably correct the IVS40 mutation within thegenomic locus of the USH2A gene. Any CRISPR endonuclease can be used inthe methods of the present disclosure, each CRISPR endonuclease havingits own associated PAM, which can or cannot be disease specific. Forexample, gRNA spacer sequences for targeting the IVS40 mutation in theUSH2A gene with a CRISPR/Cas9 endonuclease from S. pyogenes have beenidentified in SEQ ID NOs: 5272-5319, 5321, 5323, 5325, 5327-5328, and5443 of the Sequence Listing. For example, gRNA spacer sequences fortargeting the IVS40 mutation in the USH2A gene with a CRISPR/Cas9endonuclease from S. aureus have been identified in SEQ ID NOs:5446-5461 of the Sequence Listing.

Examples set forth in the present disclosure can induce single-strandedbreaks or double-stranded breaks within or near, upstream and downstreamof the IVS40 mutation within intron 40 of the USH2A gene to introducedisruption of RNA splicing consensus sequence, an excision, or correctthe IVS40 mutation within the USH2A gene with as few as a singletreatment (rather than deliver potential therapies for the lifetime ofthe patient).

Usher Syndrome

Usher syndrome is an autosomal recessive disease, characterized bysensorineural hearing loss, retinitis pigmentosa (RP) and in some cases,vestibular dysfunction. The prevalence of Usher Syndrome has beenestimated to be between 1/6000 and 1/25000.

Usher Syndrome is a clinically and genetically heterogeneous disease,accounting for about half of all cases of combined hereditarydeafness-blindness. To date the disease has been associated with 13genes. Three clinical forms of the disease have been identified (USH I,II, and III) based on the severity of the hearing impairment, thepresence or absence of vestibular dysfunction, and the age of onset ofthe disease.

Usher Syndrome Type II is the most frequent clinical form accounting forapproximately 50% of all Usher Syndrome cases. Usher Syndrome Type II ischaracterized by congenital hearing loss and progressive vision lossstarting in adolescence or adulthood. The hearing loss ranges from mildto severe and mainly affects the ability to hear high-frequency sounds.Vision loss occurs as the light-sensing cells of the retina graduallydeteriorate. Night vision loss begins first, followed by loss of theperipheral vision. With time, these blind areas enlarge and merge toproduce tunnel vision. In some cases, vision is further impaired bycataracts. Many patients become legally blind in the 5^(th) decade oflife.

Usher Syndrome Type 2A is due to a mutation in the USH2A gene andaccounts for approximately 80% of all Usher Syndrome Type II cases and40% of all Usher Syndrome cases.

USH2A Gene

The USH2A gene (e.g., SEQ ID NO: 5266) is 800,503 base pairs and islocated on Chromosome 1: 215,622,893:216,423,395 (Genome ReferenceConsortium—GRCh38/hg38) (1q41). USH2A gene comprises 72 exons andencodes for two alternatively spliced isoforms of a protein calledusherin. The full-length 580 kDa usherin protein (isoform b) is acomplex transmembrane protein of 5,202 amino acids with a largeextracellular domain. The short 170 kDa usherin protein (isoform a) istranslated from the splice variant consisting of only the first 21coding exons, and is regarded as an extracellular protein of 1546 aminoacids.

The usherin protein is located next to vesicle loading point at thepericiliary membrane and may play a role in vesicle transport betweenthe inner segments and the outer segments of photoreceptors.

IVS40 Mutation

There are various mutations associated with Usher Syndrome, which can beinsertions, deletions, missense, nonsense, frameshift and othermutations, with the common effect of inactivating the USH2A gene.

The C.7595-2144A>G (IVS40 mutation) in the USH2A gene leads to thecreation of a splice donor site and insertion of 152 bp into the USH2AmRNA, which in turn leads to a frameshift and a truncated andnon-functional protein.

Any one or more of the mutations can be repaired in order to restore theusherin protein function. For example, the pathological variant, IVS40,can be excised or corrected to restore the usherin protein expression(See Table 1).

TABLE 1 Variant Location Variant type IVS40 Chr1: 215891198 missense(GRCh38/hg38)

In Vivo Based Therapy

Provided herein are methods for treating a patient with Usher SyndromeType 2A. In some aspects, the method is an in vivo cell-based therapy.Chromosomal DNA of the cells in the Usher Syndrome type 2A patient canbe edited using the materials and methods described herein. For example,the in-vivo method can comprise editing an IVS40 mutation in a USH2Agene in a cell of a patient, such as photoreceptor cells or retinalprogenitor cells.

Although certain cells present an attractive target for ex vivotreatment and therapy, increased efficacy in delivery may permit directin vivo delivery to such cells. Ideally the targeting and editing wouldbe directed to the relevant cells. Cleavage in other cells can also beprevented by the use of promoters only active in certain cells and ordevelopmental stages. Additional promoters are inducible, and thereforecan be temporally controlled if the nuclease is delivered as a plasmid.The amount of time that delivered RNA and protein remain in the cell canalso be adjusted using treatments or domains added to change thehalf-life. In vivo treatment would eliminate a number of treatmentsteps, but a lower rate of delivery can require higher rates of editing.In vivo treatment can eliminate problems and losses from ex vivotreatment and engraftment.

An advantage of in vivo gene therapy can be the ease of therapeuticproduction and administration. The same therapeutic approach and therapywill have the potential to be used to treat more than one patient, forexample a number of patients who share the same or similar genotype orallele. In contrast, ex vivo cell therapy typically requires using apatient's own cells, which are isolated, manipulated and returned to thesame patient.

Ex Vivo Based Therapy

Provided herein are methods for treating a patient with Usher Syndrometype 2A. An aspect of such method is an ex vivo cell-based therapy. Forexample, a patient-specific induced pluripotent stem cell (iPSC) can becreated. Then, the chromosomal DNA of these iPSC cells can be editedusing the materials and methods described herein. For example, themethod can comprise editing within or near an IVS40 mutation in a USH2Agene of the iPSC. Next, the genome-edited iPSCs can be differentiatedinto other cells, such as photoreceptor cells or retinal progenitorcells. Finally, the differentiated cells, such as photoreceptor cell orretinal progenitor cell, can be implanted into the patient (i.e.,implanted into the patient's eye).

Another aspect of such method is an ex vivo cell-based therapy. Forexample, photoreceptor cells or retinal progenitor cells can be isolatedfrom the patient. Next, the chromosomal DNA of these photoreceptor cellsor retinal progenitor cells can be edited using the materials andmethods described herein. For example, the method can comprise editingwithin or near an IVS40 mutation in a USH2A gene of the photoreceptorcells or retinal progenitor cells. Finally, the genome-editedphotoreceptor cells or retinal progenitor cells can be implanted intothe patient (i.e., implanted into the patient's eye).

Another aspect of such method is an ex vivo cell-based therapy. Forexample, a mesenchymal stem cell can be isolated from the patient, whichcan be isolated from the patient's bone marrow, peripheral blood,adipose tissue, or umbilical cord. Next, the chromosomal DNA of thesemesenchymal stem cells can be edited using the materials and methodsdescribed herein. For example, the method can comprise editing within ornear an IVS40 mutation in a USH2A gene of the mesenchymal stem cells.Next, the genome-edited mesenchymal stem cells can be differentiatedinto any type of cell, e.g., photoreceptor cells or retinal progenitorcells. Finally, the differentiated cells, e.g., photoreceptor cells orretinal progenitor cells can be implanted into the patient (i.e.,implanted into the patient's eye).

One advantage of an ex vivo cell therapy approach is the ability toconduct a comprehensive analysis of the therapeutic prior toadministration. Nuclease-based therapeutics can have some level ofoff-target effects. Performing gene correction ex vivo allows one tocharacterize the corrected cell population prior to implantation. Thepresent disclosure includes sequencing the entire genome of thecorrected cells to ensure that the off-target effects, if any, can be ingenomic locations associated with minimal risk to the patient.Furthermore, populations of specific cells, including clonalpopulations, can be isolated prior to implantation.

Another advantage of ex vivo cell therapy relates to genetic correctionin iPSCs compared to other primary cell sources. iPSCs are prolific,making it easy to obtain the large number of cells that will be requiredfor a cell-based therapy. Furthermore, iPSCs are an ideal cell type forperforming clonal isolations. This allows screening for the correctgenomic correction, without risking a decrease in viability. Incontrast, other primary cells, such as photoreceptor cells or retinalprogenitor cells, are viable for only a few passages and difficult toclonally expand. Thus, manipulation of iPSCs for the treatment of UsherSyndrome Type 2A can be much easier, and can shorten the amount of timeneeded to make the desired genetic correction.

Genome Editing

Genome editing refers to the process of modifying the nucleotidesequence of a genome, such as in a precise or pre-determined manner.Examples of methods of genome editing described herein include methodsof using site-directed nucleases to cut DNA at precise target locationsin the genome, thereby creating single-strand or double-strand DNAbreaks at particular locations within the genome. Such breaks can be andregularly are repaired by natural, endogenous cellular processes, suchas HDR and NHEJ. These two main DNA repair processes consist of a familyof alternative pathways. NHEJ directly joins the DNA ends resulting froma double-strand break, sometimes with the loss or addition of nucleotidesequence, which may disrupt or enhance gene expression. HDR utilizes ahomologous sequence, or donor sequence, as a template for inserting adefined DNA sequence at the break point. The homologous sequence can bein the endogenous genome, such as a sister chromatid. Alternatively, thedonor can be an exogenous nucleic acid, such as a plasmid, asingle-strand oligonucleotide, a double-stranded oligonucleotide, aduplex oligonucleotide or a virus, that has regions of high homologywith the nuclease-cleaved locus, but which can also contain additionalsequence or sequence changes including deletions that can beincorporated into the cleaved target locus. A third repair mechanism canbe microhomology-mediated end joining (MMEJ), also referred to as“Alternative NHEJ (ANHEJ)”, in which the genetic outcome is similar toNHEJ in that small deletions and insertions can occur at the cleavagesite. MMEJ can make use of homologous sequences of a few base pairsflanking the DNA break site to drive a more favored DNA end joiningrepair outcome, and recent reports have further elucidated the molecularmechanism of this process. In some instances, it may be possible topredict likely repair outcomes based on analysis of potentialmicrohomologies at the site of the DNA break.

Each of these genome editing mechanisms can be used to create desiredgenomic alterations. A step in the genome editing process can be tocreate one or two DNA breaks, the latter as double-strand breaks or astwo single-stranded breaks, in the target locus as near the site ofintended mutation. This can be achieved via the use of site-directedpolypeptides, as described and illustrated herein.

Site-directed polypeptides, such as a DNA endonuclease, can introducedouble-strand breaks or single-strand breaks in nucleic acids, e.g.,genomic DNA. The double-strand break can stimulate a cell's endogenousDNA-repair pathways [e.g., homology-dependent repair (HDR) ornon-homologous end joining (NHEJ) or (ANHEJ) or (MMEJ)]. NHEJ can repaircleaved target nucleic acid without the need for a homologous template.This can sometimes result in small deletions or insertions (indels) inthe target nucleic acid at the site of cleavage, and can lead todisruption or alteration of gene expression.

HDR can occur when a homologous repair template, or donor, is available.The homologous donor template can comprise at least a portion of thewild-type USH2A gene, or cDNA. The at least a portion of the wild-typeUSH2A gene or cDNA can be exon 1, exon 2, exon 3, exon 4, exon 5, exon6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14,exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22,exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30,exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38,exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46,exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54,exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62,exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70,exon 71, exon 72, intronic regions, fragments or combinations thereof,or the entire USH2A gene or cDNA.

The donor template can be either a single or double-strandedpolynucleotide. The donor template can be up to 11 kb. The donortemplate can be up to 10 kb. The donor template can be up to 9 kb. Thedonor template can be up to 8 kb. The donor template can be up to 7 kb.The donor template can be up to 6 kb. The donor template can be up to 5kb. The donor template can be up to 4 kb. The donor template can be upto 3 kb. The donor template can be up to 2 kb. The donor template can beup to 1 kb. The donor template can be less than 1 kb. The donor templatecan be 500 bp to 1000 bp. The donor template can be 250 bp to 500 bp.The donor template can be 100 to 250 bp. The donor template can bedelivered by AAV. The homologous donor template can comprise sequencesthat can be homologous to sequences flanking the target nucleic acidcleavage site. For example, the donor template can have homologous armsto the 1q41 region. The donor template can also have homologous arms tothe pathological variant IVS40. The sister chromatid can be used by thecell as the repair template. However, for the purposes of genomeediting, the repair template can be supplied as an exogenous nucleicacid, such as a plasmid, duplex oligonucleotide, single-strandoligonucleotide, double-stranded oligonucleotide, or viral nucleic acid.With exogenous donor templates, an additional nucleic acid sequence(such as a transgene) or modification (such as a single or multiple basechange or a deletion) can be introduced between the flanking regions ofhomology so that the additional or altered nucleic acid sequence alsobecomes incorporated into the target locus. MMEJ can result in a geneticoutcome that is similar to NHEJ in that small deletions and insertionscan occur at the cleavage site. MMEJ can make use of homologoussequences of a few base pairs flanking the cleavage site to drive afavored end-joining DNA repair outcome. In some instances, it may bepossible to predict likely repair outcomes based on analysis ofpotential microhomologies in the nuclease target regions.

Thus, in some cases, homologous recombination can be used to insert anexogenous polynucleotide sequence into the target nucleic acid cleavagesite. An exogenous polynucleotide sequence is termed a donorpolynucleotide (or donor or donor sequence or polynucleotide donortemplate) herein. The donor polynucleotide, a portion of the donorpolynucleotide, a copy of the donor polynucleotide, or a portion of acopy of the donor polynucleotide can be inserted into the target nucleicacid cleavage site. The donor polynucleotide can be an exogenouspolynucleotide sequence, i.e., a sequence that does not naturally occurat the target nucleic acid cleavage site.

The modifications of the target DNA due to NHEJ and/or HDR can lead to,for example, gene correction.

CRISPR Endonuclease System

A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)genomic locus can be found in the genomes of many prokaryotes (e.g.,bacteria and archaea). In prokaryotes, the CRISPR locus encodes productsthat function as a type of immune system to help defend the prokaryotesagainst foreign invaders, such as virus and phage. There are threestages of CRISPR locus function: integration of new sequences into theCRISPR locus, expression of CRISPR RNA (crRNA), and silencing of foreigninvader nucleic acid. Five types of CRISPR systems (e.g., Type I, TypeII, Type III, Type U, and Type V) have been identified.

A CRISPR locus includes a number of short repeating sequences referredto as “repeats.” When expressed, the repeats can form secondarystructures (e.g., hairpins) and/or comprise unstructured single-strandedsequences. The repeats usually occur in clusters and frequently divergebetween species. The repeats are regularly interspaced with uniqueintervening sequences referred to as “spacers,” resulting in arepeat-spacer-repeat locus architecture. The spacers are identical to orhave high homology with known foreign invader sequences. A spacer-repeatunit encodes a crisprRNA (crRNA), which is processed into a mature formof the spacer-repeat unit. A crRNA comprises a “seed” or spacer sequencethat is involved in targeting a target nucleic acid (in the naturallyoccurring form in prokaryotes, the spacer sequence targets the foreigninvader nucleic acid). A spacer sequence is located at the 5′ or 3′ endof the crRNA.

A CRISPR locus also comprises polynucleotide sequences encoding CRISPRAssociated (Cas) genes. Cas genes encode endonucleases involved in thebiogenesis and the interference stages of crRNA function in prokaryotes.Some Cas genes comprise homologous secondary and/or tertiary structures.

Type II CRISPR Systems

crRNA biogenesis in a Type II CRISPR system in nature requires atrans-activating CRISPR RNA (tracrRNA). The tracrRNA can be modified byendogenous RNaselll, and then hybridizes to a crRNA repeat in thepre-crRNA array. Endogenous RNaselll can be recruited to cleave thepre-crRNA. Cleaved crRNAs can be subjected to exoribonuclease trimmingto produce the mature crRNA form (e.g., 5′ trimming). The tracrRNA canremain hybridized to the crRNA, and the tracrRNA and the crRNA associatewith a site-directed polypeptide (e.g., Cas9). The crRNA of thecrRNA-tracrRNA-Cas9 complex can guide the complex to a target nucleicacid to which the crRNA can hybridize. Hybridization of the crRNA to thetarget nucleic acid can activate Cas9 for targeted nucleic acidcleavage. The target nucleic acid in a Type II CRISPR system is referredto as a protospacer adjacent motif (PAM). In nature, the PAM isessential to facilitate binding of a site-directed polypeptide (e.g.,Cas9) to the target nucleic acid. Type II systems (also referred to asNmeni or CASS4) are further subdivided into Type II-A (CASS4) and II-B(CASS4a). Jinek et al., Science, 337(6096):816-821 (2012) showed thatthe CRISPR/Cas9 system is useful for RNA-programmable genome editing,and international patent application publication number WO2013/176772provides numerous examples and applications of the CRISPR/Casendonuclease system for site-specific gene editing.

Type V CRISPR Systems

Type V CRISPR systems have several important differences from Type IIsystems. For example, Cpf1 is a single RNA-guided endonuclease that, incontrast to Type II systems, lacks tracrRNA. In fact, Cpf1-associatedCRISPR arrays can be processed into mature crRNAs without therequirement of an additional trans-activating tracrRNA. The Type VCRISPR array can be processed into short mature crRNAs of 42-44nucleotides in length, with each mature crRNA beginning with 19nucleotides of direct repeat followed by 23-25 nucleotides of spacersequence. In contrast, mature crRNAs in Type II systems can start with20-24 nucleotides of spacer sequence followed by about 22 nucleotides ofdirect repeat. Also, Cpf1 can utilize a T-rich protospacer-adjacentmotif such that Cpf1-crRNA complexes efficiently cleave target DNApreceded by a short T-rich PAM, which is in contrast to the G-rich PAMfollowing the target DNA for Type II systems. Thus, Type V systemscleave at a point that is distant from the PAM, while Type II systemscleave at a point that is adjacent to the PAM. In addition, in contrastto Type II systems, Cpf1 cleaves DNA via a staggered DNA double-strandedbreak with a 4 or 5 nucleotide 5′ overhang. Type II systems cleave via ablunt double-stranded break. Similar to Type II systems, Cpf1 contains apredicted RuvC-like endonuclease domain, but lacks a second HNHendonuclease domain, which is in contrast to Type II systems.

Cas Genes/Polypeptides and Protospacer Adjacent Motifs

Exemplary CRISPR/Cas polypeptides include the Cas9 polypeptides in FIG.1 of Fonfara et al., Nucleic Acids Research, 42: 2577-2590 (2014). TheCRISPR/Cas gene naming system has undergone extensive rewriting sincethe Cas genes were discovered. FIG. 5 of Fonfara, supra, provides PAMsequences for the Cas9 polypeptides from various species.

Site-Directed Polypeptides

A site-directed polypeptide is a nuclease used in genome editing tocleave DNA. The site-directed nuclease can be administered to a cell ora patient as either: one or more polypeptides, or one or more mRNAsencoding the polypeptide. Any of the enzymes or orthologs listed in SEQID NOs: 1-612, or disclosed herein, can be utilized in the methodsherein.

In the context of a CRISPR/Cas or CRISPR/Cpf1 system, the site-directedpolypeptide can bind to a guide RNA that, in turn, specifies the site inthe target DNA to which the polypeptide is directed. In the CRISPR/Casor CRISPR/Cpf1 systems disclosed herein, the site-directed polypeptidecan be an endonuclease, such as a DNA endonuclease.

A site-directed polypeptide can comprise a plurality of nucleicacid-cleaving (i.e., nuclease) domains. Two or more nucleicacid-cleaving domains can be linked together via a linker. For example,the linker can comprise a flexible linker. Linkers can comprise 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40 or more amino acids in length.

Naturally-occurring wild-type Cas9 enzymes comprise two nucleasedomains, a HNH nuclease domain and a RuvC domain. Herein, the “Cas9”refers to both naturally occurring and recombinant Cas9s. Cas9 enzymescontemplated herein can comprise a HNH or HNH-like nuclease domain,and/or a RuvC or RuvC-like nuclease domain.

HNH or HNH-like domains comprise a McrA-like fold. HNH or HNH-likedomains comprises two antiparallel β-strands and an α-helix. HNH orHNH-like domains comprises a metal binding site (e.g., a divalent cationbinding site). HNH or HNH-like domains can cleave one strand of a targetnucleic acid (e.g., the complementary strand of the crRNA targetedstrand).

RuvC or RuvC-like domains comprise an RNaseH or RNaseH-like fold.

RuvC/RNaseH domains are involved in a diverse set of nucleic acid-basedfunctions including acting on both RNA and DNA. The RNaseH domaincomprises 5 β-strands surrounded by a plurality of α-helices.RuvC/RNaseH or RuvC/RNaseH-like domains comprise a metal binding site(e.g., a divalent cation binding site). RuvC/RNaseH or RuvC/RNaseH-likedomains can cleave one strand of a target nucleic acid (e.g., thenon-complementary strand of a double-stranded target DNA).

Site-directed polypeptides can introduce double-strand breaks orsingle-strand breaks in nucleic acids, e.g., genomic DNA. Thedouble-strand break can stimulate a cell's endogenous DNA-repairpathways (e.g., HDR or NHEJ or ANHEJ or MMEJ). NHEJ can repair cleavedtarget nucleic acid without the need for a homologous template. This cansometimes result in small deletions or insertions (indels) in the targetnucleic acid at the site of cleavage, and can lead to disruption oralteration of gene expression. HDR can occur when a homologous repairtemplate, or donor, is available. The homologous donor template cancomprise sequences that are homologous to sequences flanking the targetnucleic acid cleavage site. The sister chromatid can be used by the cellas the repair template. However, for the purposes of genome editing, therepair template can be supplied as an exogenous nucleic acid, such as aplasmid, duplex oligonucleotide, single-strand oligonucleotide or viralnucleic acid. With exogenous donor templates, an additional nucleic acidsequence (such as a transgene) or modification (such as a single ormultiple base change or a deletion) can be introduced between theflanking regions of homology so that the additional or altered nucleicacid sequence also becomes incorporated into the target locus. MMEJ canresult in a genetic outcome that is similar to NHEJ in that smalldeletions and insertions can occur at the cleavage site. MMEJ can makeuse of homologous sequences of a few base pairs flanking the cleavagesite to drive a favored end-joining DNA repair outcome. In someinstances, it may be possible to predict likely repair outcomes based onanalysis of potential microhomologies in the nuclease target regions.

Thus, in some cases, homologous recombination can be used to insert anexogenous polynucleotide sequence into the target nucleic acid cleavagesite. An exogenous polynucleotide sequence is termed a donorpolynucleotide (or donor or donor sequence) herein. The donorpolynucleotide, a portion of the donor polynucleotide, a copy of thedonor polynucleotide, or a portion of a copy of the donor polynucleotidecan be inserted into the target nucleic acid cleavage site. The donorpolynucleotide can be an exogenous polynucleotide sequence, i.e., asequence that does not naturally occur at the target nucleic acidcleavage site.

The site-directed polypeptide can comprise an amino acid sequence havingat least 10%, at least 15%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%, or 100% amino acidsequence identity to a wild-type exemplary site-directed polypeptide[e.g., Cas9 from S. pyogenes, US2014/0068797 Sequence ID No. 8 orSapranauskas et al., Nucleic Acids Res, 39(21): 9275-9282 (2011)], andvarious other site-directed polypeptides. The site-directed polypeptidecan comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identityto a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes,supra) over 10 contiguous amino acids. The site-directed polypeptide cancomprise at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to awild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra)over 10 contiguous amino acids. The site-directed polypeptide cancomprise at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to awild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra)over 10 contiguous amino acids in a HNH nuclease domain of thesite-directed polypeptide. The site-directed polypeptide can comprise atmost: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-typesite-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10contiguous amino acids in a HNH nuclease domain of the site-directedpolypeptide. The site-directed polypeptide can comprise at least: 70,75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-typesite-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10contiguous amino acids in a RuvC nuclease domain of the site-directedpolypeptide. The site-directed polypeptide can comprise at most: 70, 75,80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directedpolypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguousamino acids in a RuvC nuclease domain of the site-directed polypeptide.

The site-directed polypeptide can comprise a modified form of awild-type exemplary site-directed polypeptide. The modified form of thewild-type exemplary site-directed polypeptide can comprise a mutationthat reduces the nucleic acid-cleaving activity of the site-directedpolypeptide. The modified form of the wild-type exemplary site-directedpolypeptide can have less than 90%, less than 80%, less than 70%, lessthan 60%, less than 50%, less than 40%, less than 30%, less than 20%,less than 10%, less than 5%, or less than 1% of the nucleicacid-cleaving activity of the wild-type exemplary site-directedpolypeptide (e.g., Cas9 from S. pyogenes, supra). The modified form ofthe site-directed polypeptide can have no substantial nucleicacid-cleaving activity. When a site-directed polypeptide is a modifiedform that has no substantial nucleic acid-cleaving activity, it isreferred to herein as “enzymatically inactive.”

The modified form of the site-directed polypeptide can comprise amutation such that it can induce a SSB on a target nucleic acid (e.g.,by cutting only one of the sugar-phosphate backbones of a double-strandtarget nucleic acid). The mutation can result in less than 90%, lessthan 80%, less than 70%, less than 60%, less than 50%, less than 40%,less than 30%, less than 20%, less than 10%, less than 5%, or less than1% of the nucleic acid-cleaving activity in one or more of the pluralityof nucleic acid-cleaving domains of the wild-type site directedpolypeptide (e.g., Cas9 from S. pyogenes, supra). The mutation canresult in one or more of the plurality of nucleic acid-cleaving domainsretaining the ability to cleave the complementary strand of the targetnucleic acid, but reducing its ability to cleave the non-complementarystrand of the target nucleic acid. The mutation can result in one ormore of the plurality of nucleic acid-cleaving domains retaining theability to cleave the non-complementary strand of the target nucleicacid, but reducing its ability to cleave the complementary strand of thetarget nucleic acid. For example, residues in the wild-type exemplary S.pyogenes Cas9 polypeptide, such as Asp10, His840, Asn854 and Asn856, aremutated to inactivate one or more of the plurality of nucleicacid-cleaving domains (e.g., nuclease domains). The residues to bemutated can correspond to residues Asp10, His840, Asn854 and Asn856 inthe wild-type exemplary S. pyogenes Cas9 polypeptide (e.g., asdetermined by sequence and/or structural alignment). Non-limitingexamples of mutations include D10A, H840A, N854A or N856A. Mutationsother than alanine substitutions can be suitable.

A D10A mutation can be combined with one or more of H840A, N854A, orN856A mutations to produce a site-directed polypeptide substantiallylacking DNA cleavage activity. A H840A mutation can be combined with oneor more of D10A, N854A, or N856A mutations to produce a site-directedpolypeptide substantially lacking DNA cleavage activity. A N854Amutation can be combined with one or more of H840A, D10A, or N856Amutations to produce a site-directed polypeptide substantially lackingDNA cleavage activity. A N856A mutation can be combined with one or moreof H840A, N854A, or D10A mutations to produce a site-directedpolypeptide substantially lacking DNA cleavage activity. Site-directedpolypeptides that comprise one substantially inactive nuclease domainare referred to as “nickases”.

Nickase variants of RNA-guided endonucleases, for example Cas9, can beused to increase the specificity of CRISPR-mediated genome editing. Wildtype Cas9 is typically guided by a single guide RNA designed tohybridize with a specified ˜20 nucleotide sequence in the targetsequence (such as an endogenous genomic locus). However, severalmismatches can be tolerated between the guide RNA and the target locus,effectively reducing the length of required homology in the target siteto, for example, as little as 13 nt of homology, and thereby resultingin elevated potential for binding and double-strand nucleic acidcleavage by the CRISPR/Cas9 complex elsewhere in the target genome—alsoknown as off-target cleavage. Because nickase variants of Cas9 each onlycut one strand, in order to create a double-strand break it is necessaryfor a pair of nickases to bind in close proximity and on oppositestrands of the target nucleic acid, thereby creating a pair of nicks,which is the equivalent of a double-strand break. This requires that twoseparate guide RNAs—one for each nickase—must bind in close proximityand on opposite strands of the target nucleic acid. This requirementessentially doubles the minimum length of homology needed for thedouble-strand break to occur, thereby reducing the likelihood that adouble-strand cleavage event will occur elsewhere in the genome, wherethe two guide RNA sites—if they exist—are unlikely to be sufficientlyclose to each other to enable the double-strand break to form. Asdescribed in the art, nickases can also be used to promote HDR versusNHEJ. HDR can be used to introduce selected changes into target sites inthe genome through the use of specific donor sequences that effectivelymediate the desired changes.

Mutations contemplated can include substitutions, additions, anddeletions, or any combination thereof. The mutation converts the mutatedamino acid to alanine. The mutation converts the mutated amino acid toanother amino acid (e.g., glycine, serine, threonine, cysteine, valine,leucine, isoleucine, methionine, proline, phenylalanine, tyrosine,tryptophan, aspartic acid, glutamic acid, asparagine, glutamine,histidine, lysine, or arginine). The mutation converts the mutated aminoacid to a non-natural amino acid (e.g., selenomethionine). The mutationconverts the mutated amino acid to amino acid mimics (e.g.,phosphomimics). The mutation can be a conservative mutation. Forexample, the mutation converts the mutated amino acid to amino acidsthat resemble the size, shape, charge, polarity, conformation, and/orrotamers of the mutated amino acids (e.g., cysteine/serine mutation,lysine/asparagine mutation, histidine/phenylalanine mutation). Themutation can cause a shift in reading frame and/or the creation of apremature stop codon. Mutations can cause changes to regulatory regionsof genes or loci that affect expression of one or more genes.

The site-directed polypeptide (e.g., variant, mutated, enzymaticallyinactive and/or conditionally enzymatically inactive site-directedpolypeptide) can target nucleic acid. The site-directed polypeptide(e.g., variant, mutated, enzymatically inactive and/or conditionallyenzymatically inactive endoribonuclease) can target DNA. Thesite-directed polypeptide (e.g., variant, mutated, enzymaticallyinactive and/or conditionally enzymatically inactive endoribonuclease)can target RNA.

The site-directed polypeptide can comprise one or more non-nativesequences (e.g., the site-directed polypeptide is a fusion protein).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), a nucleic acid binding domain, and two nucleic acidcleaving domains (i.e., a HNH domain and a RuvC domain).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains, wherein oneor both of the nucleic acid cleaving domains comprise at least 50% aminoacid identity to a nuclease domain from Cas9 from a bacterium (e.g., S.pyogenes).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), and non-native sequence (for example, anuclear localization signal) or a linker linking the site-directedpolypeptide to a non-native sequence.

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), wherein the site-directed polypeptidecomprises a mutation in one or both of the nucleic acid cleaving domainsthat reduces the cleaving activity of the nuclease domains by at least50%.

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), wherein one of the nuclease domains comprisesmutation of aspartic acid 10, and/or wherein one of the nuclease domainscan comprise a mutation of histidine 840, and wherein the mutationreduces the cleaving activity of the nuclease domain(s) by at least 50%.

The one or more site-directed polypeptides, e.g., DNA endonucleases, cancomprise two nickases that together effect one double-strand break at aspecific locus in the genome, or four nickases that together effect orcause two double-strand breaks at specific loci in the genome.Alternatively, one site-directed polypeptide, e.g., DNA endonuclease,can effect or cause one double-strand break at a specific locus in thegenome.

Non-limiting examples of Cas9 orthologs from other bacterial strainsincluding but not limited to, Cas proteins identified in Acaryochlorismarina MBIC11017; Acetohalobium arabaticum DSM 5501; Acidithiobacilluscaldus; Acidithiobacillus ferrooxidans ATCC 23270; Alicyclobacillusacidocaldarius LAA1; Alicyclobacillus acidocaldarius subsp.acidocaldarius DSM 446; Allochromatium vinosum DSM 180; Ammonifexdegensii KC4; Anabaena variabilis ATCC 29413; Arthrospira maxima CS-328;Arthrospira platensis str. Paraca; Arthrospira sp. PCC 8005; Bacilluspseudomycoides DSM 12442; Bacillus selenitireducens MLS10;Burkholderiales bacterium 1_1_47; Caldicelulosiruptor becscii DSM 6725;Candidatus Desulforudis audaxviator MP104C; Caldicellulosiruptorhydrothermalis_108; Clostridium phage c-st; Clostridium botulinum A3str. Loch Maree; Clostridium botulinum Ba4 str. 657; Clostridiumdifficile QCD-63q42; Crocosphaera watsonii WH 8501; Cyanothece sp. ATCC51142; Cyanothece sp. CCY0110; Cyanothece sp. PCC 7424; Cyanothece sp.PCC 7822; Exiguobacterium sibiricum 255-15; Finegoldia magna ATCC 29328;Ktedonobacter racemifer DSM 44963; Lactobacillus delbrueckii subsp.bulgaricus PB2003/044-T3-4; Lactobacillus salivarius ATCC 11741;Listeria innocua; Lyngbya sp. PCC 8106; Marinobacter sp. ELB17;Methanohalobium evestigatum Z-7303; Microcystis phage Ma-LMM01;Microcystis aeruginosa NIES-843; Microscilla marina ATCC 23134;Microcoleus chthonoplastes PCC 7420; Neisseria meningitidis;Nitrosococcus halophilus Nc4; Nocardiopsis dassonvillei subsp.dassonvillei DSM 43111; Nodularia spumigena CCY9414; Nostoc sp. PCC7120; Oscillatoria sp. PCC 6506; Pelotomaculum_thermopropionicum_SI;Petrotoga mobilis SJ95; Polaromonas naphthalenivorans CJ2; Polaromonassp. JS666; Pseudoalteromonas haloplanktis TAC125; Streptomycespristinaespiralis ATCC 25486; Streptomyces pristinaespiralis ATCC 25486;Streptococcus thermophilus; Streptomyces viridochromogenes DSM 40736;Streptosporangium roseum DSM 43021; Synechococcus sp. PCC 7335; andThermosipho africanus TCF52B (Chylinski et al., RNA Biol., 2013; 10(5):726-737.

In addition to Cas9 orthologs, other Cas9 variants such as fusionproteins of inactive dCas9 and effector domains with different functionscan be served as a platform for genetic modulation. Any of the foregoingenzymes can be useful in the present disclosure.

Further examples of endonucleases that can be utilized in the presentdisclosure are provided in SEQ ID NOs: 1-612. These proteins can bemodified before use or can be encoded in a nucleic acid sequence such asa DNA, RNA or mRNA or within a vector construct such as the plasmids oradeno-associated virus (AAV) vectors taught herein. Further, they can becodon optimized.

Although nomenclature is used herein to indicate the species of originfor a given site-directed polypeptide, it is understood that thesite-directed polypeptide and/or the nucleic acid encoding thesite-directed polypeptide can be modified compared to the sequenceoccurring in the species of origin. For example, “SpCas9” indicates thatthe Cas9 gene/protein in question originated in Streptococcus pyogenesand was modified, such as by addition of NLS(s) and/or the performanceof codon optimization. For example, “SaCas9” indicates that the Cas9gene/protein in question originated in Staphylococcus aureus and wasmodified, such as by addition of NLS(s) and/or the performance of codonoptimization.

Genome-Targeting Nucleic Acid

The present disclosure provides a genome-targeting nucleic acid that candirect the activities of an associated polypeptide (e.g., asite-directed polypeptide) to a specific target sequence within a targetnucleic acid. The genome-targeting nucleic acid can be an RNA. Agenome-targeting RNA is referred to as a “guide RNA” or “gRNA” herein. Aguide RNA can comprise at least a spacer sequence that hybridizes to atarget nucleic acid sequence of interest, and a CRISPR repeat sequence.In Type II systems, the gRNA also comprises a second RNA called thetracrRNA sequence. In the Type II gRNA, the CRISPR repeat sequence andtracrRNA sequence hybridize to each other to form a duplex. In the TypeV gRNA, the crRNA forms a duplex. In both systems, the duplex can bind asite-directed polypeptide, such that the guide RNA and site-directpolypeptide form a complex. The genome-targeting nucleic acid canprovide target specificity to the complex by virtue of its associationwith the site-directed polypeptide. The genome-targeting nucleic acidthus can direct the activity of the site-directed polypeptide.

Exemplary guide RNAs include the spacer sequences in SEQ ID NOs:5272-5319, 5321, 5323, 5325, 5327-5328, 5443 and 5446-5461 of theSequence Listing (FIGS. 2A-B, G, and J). The target DNA sequence (5′-3′)(See SEQ ID NOs: 5329-5385, 5444, and 5462-5477) can be found in FIGS.2C-D, 2H, and 2K. The reverse strand of target DNA sequence to which thesgRNA will bind (5′-3′) can be found in FIGS. 2E-F, I, and L.

Each guide RNA can be designed to include a spacer sequencecomplementary to its genomic target sequence. For example, each of thespacer sequences in SEQ ID NOs: 5272-5319, 5321, 5323, 5325, 5327-5328,5443 and 5446-5461 of the Sequence Listing can be put into a single RNAchimera or a crRNA (along with a corresponding tracrRNA). See Jinek etal., Science, 337, 816-821 (2012) and Deltcheva et al., Nature, 471,602-607 (2011).

gRNAs or sgRNAs disclosed herein can associate with a DNA endonucleaseto form a ribonucleoprotein complex, which can cause stable edits withinor near the IVS40 mutation in a USH2A gene. Changes in DNA sequences(e.g., edits) brought about by this editing can be non-transient.

The genome-targeting nucleic acid can be a double-molecule guide RNA(FIG. 1A). The genome-targeting nucleic acid can be a single-moleculeguide RNA (FIG. 1B). The double-molecule guide RNA or single-moleculeguide RNA can be modified.

A double-molecule guide RNA can comprise two strands of RNA. The firststrand comprises in the 5′ to 3′ direction, an optional spacer extensionsequence, a spacer sequence and a minimum CRISPR repeat sequence. Thesecond strand can comprise a minimum tracrRNA sequence (complementary tothe minimum CRISPR repeat sequence), a 3′ tracrRNA sequence and anoptional tracrRNA extension sequence.

A single-molecule guide RNA (sgRNA) in a Type II system can comprise, inthe 5′ to 3′ direction, an optional spacer extension sequence, a spacersequence, a minimum CRISPR repeat sequence, a single-molecule guidelinker, a minimum tracrRNA sequence, a 3′ tracrRNA sequence and anoptional tracrRNA extension sequence. The optional tracrRNA extensioncan comprise elements that contribute additional functionality (e.g.,stability) to the guide RNA. The single-molecule guide linker can linkthe minimum CRISPR repeat and the minimum tracrRNA sequence to form ahairpin structure. The optional tracrRNA extension can comprise one ormore hairpins.

The sgRNA can comprise a variable length spacer sequence with 17-30nucleotides at the 5′ end of the sgRNA sequence (Table 2). In otherexamples, the sgRNA can comprise a variable length spacer sequence with17-24 nucleotides at the 5′ end of the sgRNA sequence.

The sgRNA can comprise a 20 nucleotide spacer sequence at the 5′ end ofthe sgRNA sequence. The sgRNA can comprise a less than 20 nucleotidespacer sequence at the 5′ end of thesgRNA sequence. The sgRNA cancomprise a 19 nucleotide spacer sequence at the 5′ end of thesgRNAsequence. The sgRNA can comprise a 18 nucleotide spacer sequence at the5′ end of thesgRNA sequence. The sgRNA can comprise a 17 nucleotidespacer sequence at the 5′ end of thesgRNA sequence. The sgRNA cancomprise a more than 20 nucleotide spacer sequence at the 5′end of thesgRNA sequence. The sgRNA can comprise a 21 nucleotide spacer sequenceat the 5′end of the sgRNA sequence. The sgRNA can comprise a 22nucleotide spacer sequence at the 5′end of the sgRNA sequence. The sgRNAcan comprise a 23 nucleotide spacer sequence at the 5′end of the sgRNAsequence. The sgRNA can comprise a 24 nucleotide spacer sequence at the5′end of the sgRNA sequence. The sgRNA can comprise a 25 nucleotidespacer sequence at the 5′end of the sgRNA sequence. The sgRNA cancomprise a 26 nucleotide spacer sequence at the 5′end of the sgRNAsequence. The sgRNA can comprise a 27 nucleotide spacer sequence at the5′end of the sgRNA sequence. The sgRNA can comprise a 28 nucleotidespacer sequence at the 5′end of the sgRNA sequence. The sgRNA cancomprise a 29 nucleotide spacer sequence at the 5′end of the sgRNAsequence. The sgRNA can comprise a 30 nucleotide spacer sequence at the5′ end of the sgRNA sequence.

The sgRNA can comprise no uracil at the 3′end of the sgRNA sequence,such as in SEQ ID NOs: 5268, 5527, 5530, 5533, or 5536 of Table 2. ThesgRNA can comprise one or more uracil at the 3′end of the sgRNAsequence, such as in SEQ ID NOs: 5267, 5269, 5526, 5528, 5529, 5531,5532, 5534, 5535, or 5537 in Table 2. For example, the sgRNA cancomprise 1 uracil (U) at the 3′ end of the sgRNA sequence. The sgRNA cancomprise 2 uracil (UU) at the 3′ end of the sgRNA sequence. The sgRNAcan comprise 3 uracil (UUU) at the 3′ end of the sgRNA sequence. ThesgRNA can comprise 4 uracil (UUUU) at the 3′ end of the sgRNA sequence.The sgRNA can comprise 5 uracil (UUUUU) at the 3′ end of the sgRNAsequence. The sgRNA can comprise 6 uracil (UUUUUU) at the 3′ end of thesgRNA sequence. The sgRNA can comprise 7 uracil (UUUUUUU) at the 3′ endof the sgRNA sequence. The sgRNA can comprise 8 uracil (UUUUUUUU) at the3′ end of the sgRNA sequence.

The sgRNA can be unmodified or modified. For example, modified sgRNAscan comprise one or more 2′-O-methyl phosphorothioate nucleotides.

TABLE 2 SEQ ID NO. sgRNA sequence 5267n₍₁₇₋₃₀₎guuuuagagcuagaaauagcaaguuaaaauaa Spggcuaguccguuaucaacuugaaaaaguggcaccgag ucggugcuuuu 5268n₍₁₇₋₃₀₎guuuuagagcuagaaauagcaaguuaaaauaa Spggcuaguccguuaucaacuugaaaaaguggcaccgagu cggugc 5269n₍₁₇₋₃₀₎guuuuagagcuagaaauagcaaguuaaaauaa Spggcuaguccguuaucaacuugaaaaaguggcaccgagu cggugcu₍₁₋₈₎ 5526n₍₁₇₋₃₀₎guuuaaguacucugugcuggaaacagcacagaa Saucuacuuaaacaaggcaaaaugccguguuuaucucguca acuuguuggcgagauuuuuu 5527n₍₁₇₋₃₀₎guuuaaguacucugugcuggaaacagcacagaa Saucuacuuaaacaaggcaaaaugccguguuuaucucguca acuuguuggcgaga 5528n₍₁₇₋₃₀₎guuuaaguacucugugcuggaaacagcacagaa Saucuacuuaaacaaggcaaaaugccguguuuaucucguca acuuguuggcgagau₍₁₋₈₎ 5529n₍₁₇₋₃₀₎guuuuaguacucuguaaugaaaauuacagaa Saucuacuaaaacaaggcaaaaugccguguuuaucucgu caacuuguuggcgagauuuuuuuu 5530n₍₁₇₋₃₀₎guuuuaguacucuguaaugaaaauuacagaa Saucuacuaaaacaaggcaaaaugccguguuuaucucgu caacuuguuggcgaga 5531n₍₁₇₋₃₀₎guuuuaguacucuguaaugaaaauuacagaa Saucuacuaaaacaaggcaaaaugccguguuuaucucgu caacuuguuggcgagau₍₁₋₈₎ 5532n₍₁₇₋₃₀₎guuuaaguacucugugcuggaaacagcacagaa Saucuacuuaaacaaggcaaaaugccguguuuaucucguc aacuuguuggcgagauuuuuuuu 5533n₍₁₇₋₃₀₎guuuaaguacucugugcuggaaacagcacagaa Saucuacuuaaacaaggcaaaaugccguguuuaucucguc aacuuguuggcgaga 5534n₍₁₇₋₃₀₎guuuaaguacucugugcuggaaacagcacagaa Saucuacuuaaacaaggcaaaaugccguguuuaucucguc aacuuguuggcgagau₍₁₋₈₎ 5535n₍₁₇₋₃₀₎guuuuaguacucuggaaacagaaucuacuaaaa Sacaaggcaaaaugccguguuuaucucgucaacuuguugg cgagauuuu 5536n₍₁₇₋₃₀₎guuuuaguacucuggaaacagaaucuacuaaaa Sacaaggcaaaaugccguguuuaucucgucaacuuguugg cgaga 5537n₍₁₇₋₃₀₎guuuuaguacucuggaaacagaaucuacuaaaa Sacaaggcaaaaugccguguuuaucucgucaacuuguugg cgagau₍₁₋₈₎

A single-molecule guide RNA (sgRNA) in a Type V system can comprise, inthe 5′ to 3′ direction, a minimum CRISPR repeat sequence and a spacersequence.

By way of illustration, guide RNAs used in the CRISPR/Cas/Cpf1 system,or other smaller RNAs can be readily synthesized by chemical means, asillustrated below and described in the art. While chemical syntheticprocedures are continually expanding, purifications of such RNAs byprocedures such as high performance liquid chromatography (HPLC, whichavoids the use of gels such as PAGE) tends to become more challenging aspolynucleotide lengths increase significantly beyond a hundred or sonucleotides. One approach used for generating RNAs of greater length isto produce two or more molecules that are ligated together. Much longerRNAs, such as those encoding a Cas9 or Cpf1 endonuclease, are morereadily generated enzymatically. Various types of RNA modifications canbe introduced during or after chemical synthesis and/or enzymaticgeneration of RNAs, e.g., modifications that enhance stability, reducethe likelihood or degree of innate immune response, and/or enhance otherattributes, as described in the art.

Spacer Extension Sequence

In some examples of genome-targeting nucleic acids, a spacer extensionsequence can modify activity, provide stability and/or provide alocation for modifications of a genome-targeting nucleic acid. A spacerextension sequence can modify on- or off-target activity or specificity.In some examples, a spacer extension sequence can be provided. Thespacer extension sequence can have a length of more than 1, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000,4000, 5000, 6000, or 7000 or more nucleotides. The spacer extensionsequence can have a length of less than 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260,280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000,7000 or more nucleotides. The spacer extension sequence can be less than10 nucleotides in length. The spacer extension sequence can be between10-30 nucleotides in length. The spacer extension sequence can bebetween 30-70 nucleotides in length.

The spacer extension sequence can comprise another moiety (e.g., astability control sequence, an endoribonuclease binding sequence, aribozyme). The moiety can decrease or increase the stability of anucleic acid targeting nucleic acid. The moiety can be a transcriptionalterminator segment (i.e., a transcription termination sequence). Themoiety can function in a eukaryotic cell. The moiety can function in aprokaryotic cell. The moiety can function in both eukaryotic andprokaryotic cells. Non-limiting examples of suitable moieties include: a5′ cap (e.g., a 7-methylguanylate cap (m7 G)), a riboswitch sequence(e.g., to allow for regulated stability and/or regulated accessibilityby proteins and protein complexes), a sequence that forms a dsRNA duplex(i.e., a hairpin), a sequence that targets the RNA to a subcellularlocation (e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), and/or a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like).

Spacer Sequence

The spacer sequence hybridizes to a sequence in a target nucleic acid ofinterest. The spacer of a genome-targeting nucleic acid can interactwith a target nucleic acid in a sequence-specific manner viahybridization (i.e., base pairing). The nucleotide sequence of thespacer can vary depending on the sequence of the target nucleic acid ofinterest.

In a CRISPR/Cas system herein, the spacer sequence can be designed tohybridize to a target nucleic acid that is located 5′ of a PAM of theCas9 enzyme used in the system. The spacer can perfectly match thetarget sequence or can have mismatches. Each Cas9 enzyme has aparticular PAM sequence that it recognizes in a target DNA. For example,S. pyogenes recognizes in a target nucleic acid a PAM that comprises thesequence 5′-NRG-3′, where R comprises either A or G, where N is anynucleotide and N is immediately 3′ of the target nucleic acid sequencetargeted by the spacer sequence. For example, S. aureus Cas9 recognizesin a target nucleic acid a PAM that comprises the sequence 5′-NNGRRT-3′,where R comprises either A or G, where N is any nucleotide and N isimmediately 3′ of the target nucleic acid sequence targeted by thespacer sequence. In certain examples, S. aureus Cas9 recognizes in atarget nucleic acid a PAM that comprises the sequence 5′-NNGRRN-3′,where R comprises either A or G, where N is any nucleotide and the N isimmediately 3′ of the target nucleic acid sequence targeted by thespacer sequence. For example, C. jejuni recognizes in a target nucleicacid a PAM that comprises the sequence 5′-NNNNACA-3′ or 5′-NNNNACAC-3′,where N is any nucleotide and N is immediately 3′ of the target nucleicacid sequence targeted by the spacer sequence. In certain examples, C.jejuni Cas9 recognizes in a target nucleic acid a PAM that comprises thesequence 5′-NNNVRYM-3′ or 5′-NNVRYAC-3′, where V comprises either A, Gor C, where R comprises either A or G, where Y comprises either C or T,where M comprises A or C, where N is any nucleotide and the N isimmediately 3′ of the target nucleic acid sequence targeted by thespacer sequence.

The target nucleic acid sequence can comprise 20 nucleotides. The targetnucleic acid can comprise less than 20 nucleotides. The target nucleicacid can comprise more than 20 nucleotides. The target nucleic acid cancomprise at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30or more nucleotides. The target nucleic acid can comprise at most: 5,10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides.The target nucleic acid sequence can comprise 20 bases immediately 5′ ofthe first nucleotide of the PAM. For example, in a sequence comprising5′-NNNNNNNNNNNNNNNNNNNNNRG-3′ (SEQ ID NO: 5270), the target nucleic acidcan comprise the sequence that corresponds to the Ns, wherein N is anynucleotide, and the underlined NRG sequence is the S. pyogenes PAM.

The spacer sequence that hybridizes to the target nucleic acid can havea length of at least about 6 nucleotides (nt). The spacer sequence canbe at least about 6 nt, at least about 10 nt, at least about 15 nt, atleast about 18 nt, at least about 19 nt, at least about 20 nt, at leastabout 25 nt, at least about 30 nt, at least about 35 nt or at leastabout 40 nt, from about 6 nt to about 80 nt, from about 6 nt to about 50nt, from about 6 nt to about 45 nt, from about 6 nt to about 40 nt, fromabout 6 nt to about 35 nt, from about 6 nt to about 30 nt, from about 6nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt toabout 19 nt, from about 10 nt to about 50 nt, from about 10 nt to about45 nt, from about 10 nt to about 40 nt, from about 10 nt to about 35 nt,from about 10 nt to about 30 nt, from about 10 nt to about 25 nt, fromabout 10 nt to about 20 nt, from about 10 nt to about 19 nt, from about19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 ntto about 35 nt, from about 19 nt to about 40 nt, from about 19 nt toabout 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt,from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, fromabout 20 nt to about 45 nt, from about 20 nt to about 50 nt, or fromabout 20 nt to about 60 nt. In some examples, the spacer sequence cancomprise 24 nucleotides. In some examples, the spacer sequence cancomprise 23 nucleotides. In some examples, the spacer sequence cancomprise 22 nucleotides. In some examples, the spacer sequence cancomprise 21 nucleotides. In some examples, the spacer sequence cancomprise 20 nucleotides. In some examples, the spacer sequence cancomprise 19 nucleotides. In some examples, the spacer sequence cancomprise 18 nucleotides. In some examples, the spacer sequence cancomprise 17 nucleotides.

In some examples, the percent complementarity between the spacersequence and the target nucleic acid is at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, at least about 99%, or 100%. In some examples, thepercent complementarity between the spacer sequence and the targetnucleic acid is at most about 30%, at most about 40%, at most about 50%,at most about 60%, at most about 65%, at most about 70%, at most about75%, at most about 80%, at most about 85%, at most about 90%, at mostabout 95%, at most about 97%, at most about 98%, at most about 99%, or100%. In some examples, the percent complementarity between the spacersequence and the target nucleic acid is 100% over the six contiguous5′-most nucleotides of the target sequence of the complementary strandof the target nucleic acid. The percent complementarity between thespacer sequence and the target nucleic acid can be at least 60% overabout 20 contiguous nucleotides. The length of the spacer sequence andthe target nucleic acid can differ by 1 to 6 nucleotides, which can bethought of as a bulge or bulges.

The spacer sequence can be designed or chosen using a computer program.The computer program can use variables, such as predicted meltingtemperature, secondary structure formation, predicted annealingtemperature, sequence identity, genomic context, chromatinaccessibility, % GC, frequency of genomic occurrence (e.g., of sequencesthat are identical or are similar but vary in one or more spots as aresult of mismatch, insertion or deletion), methylation status, presenceof SNPs, and the like.

Minimum CRISPR Repeat Sequence

A minimum CRISPR repeat sequence can be a sequence with at least about30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95%, or 100% sequence identity toa reference CRISPR repeat sequence (e.g., crRNA from S. pyogenes).

A minimum CRISPR repeat sequence can comprise nucleotides that canhybridize to a minimum tracrRNA sequence in a cell. The minimum CRISPRrepeat sequence and a minimum tracrRNA sequence can form a duplex, i.e.a base-paired double-stranded structure. Together, the minimum CRISPRrepeat sequence and the minimum tracrRNA sequence can bind to thesite-directed polypeptide. At least a part of the minimum CRISPR repeatsequence can hybridize to the minimum tracrRNA sequence. At least a partof the minimum CRISPR repeat sequence can comprise at least about 30%,about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or 100% complementary to theminimum tracrRNA sequence. At least a part of the minimum CRISPR repeatsequence can comprise at most about 30%, about 40%, about 50%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or 100% complementary to the minimum tracrRNA sequence.

The minimum CRISPR repeat sequence can have a length from about 7nucleotides to about 100 nucleotides. For example, the length of theminimum CRISPR repeat sequence is from about 7 nucleotides (nt) to about50 nt, from about 7 nt to about 40 nt, from about 7 nt to about 30 nt,from about 7 nt to about 25 nt, from about 7 nt to about 20 nt, fromabout 7 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8nt to about 30 nt, from about 8 nt to about 25 nt, from about 8 nt toabout 20 nt, from about 8 nt to about 15 nt, from about 15 nt to about100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt, orfrom about 15 nt to about 25 nt. In some examples, the minimum CRISPRrepeat sequence can be approximately 9 nucleotides in length. Theminimum CRISPR repeat sequence can be approximately 12 nucleotides inlength.

The minimum CRISPR repeat sequence can be at least about 60% identicalto a reference minimum CRISPR repeat sequence (e.g., wild-type crRNAfrom S. pyogenes) over a stretch of at least 6, 7, or 8 contiguousnucleotides. For example, the minimum CRISPR repeat sequence can be atleast about 65% identical, at least about 70% identical, at least about75% identical, at least about 80% identical, at least about 85%identical, at least about 90% identical, at least about 95% identical,at least about 98% identical, at least about 99% identical or 100%identical to a reference minimum CRISPR repeat sequence over a stretchof at least 6, 7, or 8 contiguous nucleotides.

Minimum tracrRNA Sequence

A minimum tracrRNA sequence can be a sequence with at least about 30%,about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or 100% sequence identity to areference tracrRNA sequence (e.g., wild type tracrRNA from S. pyogenes).

A minimum tracrRNA sequence can comprise nucleotides that hybridize to aminimum CRISPR repeat sequence in a cell. A minimum tracrRNA sequenceand a minimum CRISPR repeat sequence form a duplex, i.e. a base-paireddouble-stranded structure. Together, the minimum tracrRNA sequence andthe minimum CRISPR repeat can bind to a site-directed polypeptide. Atleast a part of the minimum tracrRNA sequence can hybridize to theminimum CRISPR repeat sequence. The minimum tracrRNA sequence can be atleast about 30%, about 40%, about 50%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or 100%complementary to the minimum CRISPR repeat sequence.

The minimum tracrRNA sequence can have a length from about 7 nucleotidesto about 100 nucleotides. For example, the minimum tracrRNA sequence canbe from about 7 nucleotides (nt) to about 50 nt, from about 7 nt toabout 40 nt, from about 7 nt to about 30 nt, from about 7 nt to about 25nt, from about 7 nt to about 20 nt, from about 7 nt to about 15 nt, fromabout 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt toabout 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,from about 15 nt to about 30 nt or from about 15 nt to about 25 nt long.The minimum tracrRNA sequence can be approximately 9 nucleotides inlength. The minimum tracrRNA sequence can be approximately 12nucleotides. The minimum tracrRNA can consist of tracrRNA nt 23-48described in Jinek et al., supra.

The minimum tracrRNA sequence can be at least about 60% identical to areference minimum tracrRNA (e.g., wild type, tracrRNA from S. pyogenes)sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.For example, the minimum tracrRNA sequence can be at least about 65%identical, about 70% identical, about 75% identical, about 80%identical, about 85% identical, about 90% identical, about 95%identical, about 98% identical, about 99% identical or 100% identical toa reference minimum tracrRNA sequence over a stretch of at least 6, 7,or 8 contiguous nucleotides.

The duplex between the minimum CRISPR RNA and the minimum tracrRNA cancomprise a double helix. The duplex between the minimum CRISPR RNA andthe minimum tracrRNA can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 or more nucleotides. The duplex between the minimum CRISPR RNAand the minimum tracrRNA can comprise at most about 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 or more nucleotides.

The duplex can comprise a mismatch (i.e., the two strands of the duplexare not 100% complementary). The duplex can comprise at least about 1,2, 3, 4, or 5 or mismatches. The duplex can comprise at most about 1, 2,3, 4, or 5 or mismatches. The duplex can comprise no more than 2mismatches.

Bulges

In some cases, there can be a “bulge” in the duplex between the minimumCRISPR RNA and the minimum tracrRNA. A bulge is an unpaired region ofnucleotides within the duplex. A bulge can contribute to the binding ofthe duplex to the site-directed polypeptide. The bulge can comprise, onone side of the duplex, an unpaired 5′-XXXY-3′ where X is any purine andY comprises a nucleotide that can form a wobble pair with a nucleotideon the opposite strand, and an unpaired nucleotide region on the otherside of the duplex. The number of unpaired nucleotides on the two sidesof the duplex can be different.

In one example, the bulge can comprise an unpaired purine (e.g.,adenine) on the minimum CRISPR repeat strand of the bulge. In someexamples, the bulge can comprise an unpaired 5′-AAGY-3′ of the minimumtracrRNA sequence strand of the bulge, where Y comprises a nucleotidethat can form a wobble pairing with a nucleotide on the minimum CRISPRrepeat strand.

A bulge on the minimum CRISPR repeat side of the duplex can comprise atleast 1, 2, 3, 4, or 5 or more unpaired nucleotides. A bulge on theminimum CRISPR repeat side of the duplex can comprise at most 1, 2, 3,4, or 5 or more unpaired nucleotides. A bulge on the minimum CRISPRrepeat side of the duplex can comprise 1 unpaired nucleotide.

A bulge on the minimum tracrRNA sequence side of the duplex can compriseat least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides.A bulge on the minimum tracrRNA sequence side of the duplex can compriseat most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides. Abulge on a second side of the duplex (e.g., the minimum tracrRNAsequence side of the duplex) can comprise 4 unpaired nucleotides.

A bulge can comprise at least one wobble pairing. In some examples, abulge can comprise at most one wobble pairing. A bulge can comprise atleast one purine nucleotide. A bulge can comprise at least 3 purinenucleotides. A bulge sequence can comprise at least 5 purinenucleotides. A bulge sequence can comprise at least one guaninenucleotide. In some examples, a bulge sequence can comprise at least oneadenine nucleotide.

Hairpins

In various examples, one or more hairpins can be located 3′ to theminimum tracrRNA in the 3′ tracrRNA sequence.

The hairpin can start at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,or 20 or more nucleotides 3′ from the last paired nucleotide in theminimum CRISPR repeat and minimum tracrRNA sequence duplex. The hairpincan start at most about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or morenucleotides 3′ of the last paired nucleotide in the minimum CRISPRrepeat and minimum tracrRNA sequence duplex.

The hairpin can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, or 20 or more consecutive nucleotides. The hairpin can comprise atmost about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or more consecutivenucleotides.

The hairpin can comprise a CC dinucleotide (i.e., two consecutivecytosine nucleotides).

The hairpin can comprise duplexed nucleotides (e.g., nucleotides in ahairpin, hybridized together). For example, a hairpin can comprise a CCdinucleotide that is hybridized to a GG dinucleotide in a hairpin duplexof the 3′ tracrRNA sequence.

One or more of the hairpins can interact with guide RNA-interactingregions of a site-directed polypeptide.

In some examples, there are two or more hairpins, and in other examplesthere are three or more hairpins.

3′ tracrRNA Sequence

A 3′ tracrRNA sequence can comprise a sequence with at least about 30%,about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or 100% sequence identity to areference tracrRNA sequence (e.g., a tracrRNA from S. pyogenes).

The 3′ tracrRNA sequence can have a length from about 6 nucleotides toabout 100 nucleotides. For example, the 3′ tracrRNA sequence can have alength from about 6 nucleotides (nt) to about 50 nt, from about 6 nt toabout 40 nt, from about 6 nt to about 30 nt, from about 6 nt to about 25nt, from about 6 nt to about 20 nt, from about 6 nt to about 15 nt, fromabout 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt toabout 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,from about 15 nt to about 30 nt, or from about 15 nt to about 25 nt. The3′ tracrRNA sequence can have a length of approximately 14 nucleotides.

The 3′ tracrRNA sequence can be at least about 60% identical to areference 3′ tracrRNA sequence (e.g., wild type 3′ tracrRNA sequencefrom S. pyogenes) over a stretch of at least 6, 7, or 8 contiguousnucleotides. For example, the 3′ tracrRNA sequence can be at least about60% identical, about 65% identical, about 70% identical, about 75%identical, about 80% identical, about 85% identical, about 90%identical, about 95% identical, about 98% identical, about 99%identical, or 100% identical, to a reference 3′ tracrRNA sequence (e.g.,wild type 3′ tracrRNA sequence from S. pyogenes) over a stretch of atleast 6, 7, or 8 contiguous nucleotides.

The 3′ tracrRNA sequence can comprise more than one duplexed region(e.g., hairpin, hybridized region). The 3′ tracrRNA sequence cancomprise two duplexed regions.

The 3′ tracrRNA sequence can comprise a stem loop structure. The stemloop structure in the 3′ tracrRNA can comprise at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15 or 20 or more nucleotides. The stem loop structure inthe 3′ tracrRNA can comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ormore nucleotides. The stem loop structure can comprise a functionalmoiety. For example, the stem loop structure can comprise an aptamer, aribozyme, a protein-interacting hairpin, a CRISPR array, an intron, oran exon. The stem loop structure can comprise at least about 1, 2, 3, 4,or 5 or more functional moieties. The stem loop structure can compriseat most about 1, 2, 3, 4, or 5 or more functional moieties.

The hairpin in the 3′ tracrRNA sequence can comprise a P-domain. In someexamples, the P-domain can comprise a double-stranded region in thehairpin.

tracrRNA Extension Sequence

A tracrRNA extension sequence can be provided whether the tracrRNA is inthe context of single-molecule guides or double-molecule guides. ThetracrRNA extension sequence can have a length from about 1 nucleotide toabout 400 nucleotides. The tracrRNA extension sequence can have a lengthof more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360,380, or 400 nucleotides. The tracrRNA extension sequence can have alength from about 20 to about 5000 or more nucleotides. The tracrRNAextension sequence can have a length of more than 1000 nucleotides. ThetracrRNA extension sequence can have a length of less than 1, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 or morenucleotides. The tracrRNA extension sequence can have a length of lessthan 1000 nucleotides. The tracrRNA extension sequence can comprise lessthan 10 nucleotides in length. The tracrRNA extension sequence can be10-30 nucleotides in length. The tracrRNA extension sequence can be30-70 nucleotides in length.

The tracrRNA extension sequence can comprise a functional moiety (e.g.,a stability control sequence, ribozyme, endoribonuclease bindingsequence). The functional moiety can comprise a transcriptionalterminator segment (i.e., a transcription termination sequence). Thefunctional moiety can have a total length from about 10 nucleotides (nt)to about 100 nucleotides, from about 10 nt to about 20 nt, from about 20nt to about 30 nt, from about 30 nt to about 40 nt, from about 40 nt toabout 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt,or from about 90 nt to about 100 nt, from about 15 nt to about 80 nt,from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, fromabout 15 nt to about 30 nt, or from about 15 nt to about 25 nt. Thefunctional moiety can function in a eukaryotic cell. The functionalmoiety can function in a prokaryotic cell. The functional moiety canfunction in both eukaryotic and prokaryotic cells.

Non-limiting examples of suitable tracrRNA extension functional moietiesinclude a 3′ poly-adenylated tail, a riboswitch sequence (e.g., to allowfor regulated stability and/or regulated accessibility by proteins andprotein complexes), a sequence that forms a dsRNA duplex (i.e., ahairpin), a sequence that targets the RNA to a subcellular location(e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), and/or a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like). The tracrRNA extension sequence cancomprise a primer binding site or a molecular index (e.g., barcodesequence). The tracrRNA extension sequence can comprise one or moreaffinity tags.

Single-Molecule Guide Linker Sequence

The linker sequence of a single-molecule guide nucleic acid can have alength from about 3 nucleotides to about 100 nucleotides. In Jinek etal., supra, for example, a simple 4 nucleotide “tetraloop” (-GAAA-) wasused, Science, 337(6096):816-821 (2012). An illustrative linker has alength from about 3 nucleotides (nt) to about 90 nt, from about 3 nt toabout 80 nt, from about 3 nt to about 70 nt, from about 3 nt to about 60nt, from about 3 nt to about 50 nt, from about 3 nt to about 40 nt, fromabout 3 nt to about 30 nt, from about 3 nt to about 20 nt, from about 3nt to about 10 nt. For example, the linker can have a length from about3 nt to about 5 nt, from about 5 nt to about 10 nt, from about 10 nt toabout 15 nt, from about 15 nt to about 20 nt, from about 20 nt to about25 nt, from about 25 nt to about 30 nt, from about 30 nt to about 35 nt,from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, fromabout 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90nt to about 100 nt. The linker of a single-molecule guide nucleic acidcan be between 4 and 40 nucleotides. The linker can be at least about100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, or 7000 or more nucleotides. The linker can be at most about100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, or 7000 or more nucleotides.

Linkers can comprise any of a variety of sequences, although in someexamples the linker will not comprise sequences that have extensiveregions of homology with other portions of the guide RNA, which mightcause intramolecular binding that could interfere with other functionalregions of the guide. In Jinek et al., supra, a simple 4 nucleotidesequence -GAAA- was used, Science, 337(6096):816-821 (2012), butnumerous other sequences, including longer sequences can likewise beused.

The linker sequence can comprise a functional moiety. For example, thelinker sequence can comprise one or more features, including an aptamer,a ribozyme, a protein-interacting hairpin, a protein binding site, aCRISPR array, an intron, or an exon. The linker sequence can comprise atleast about 1, 2, 3, 4, or 5 or more functional moieties. In someexamples, the linker sequence can comprise at most about 1, 2, 3, 4, or5 or more functional moieties.

Complexes of a Genome-Targeting Nucleic Acid and a Site-DirectedPolypeptide

A genome-targeting nucleic acid interacts with a site-directedpolypeptide (e.g., a nucleic acid-guided nuclease such as Cas9), therebyforming a complex. The genome-targeting nucleic acid guides thesite-directed polypeptide to a target nucleic acid.

Ribonucleoprotein Complexes (RNPs)

The site-directed polypeptide and genome-targeting nucleic acid can eachbe administered separately to a cell or a patient. On the other hand,the site-directed polypeptide can be pre-complexed with one or moreguide RNAs, or one or more crRNA together with a tracrRNA. Thesite-directed polypeptide can be pre-complexed with one or more sgRNA.The pre-complexed material can then be administered to a cell or apatient. Such pre-complexed material is known as a ribonucleoproteinparticle (RNP). The site-directed polypeptide in the RNP can be, forexample, a Cas9 endonuclease or a Cpf1 endonuclease. The site-directedpolypeptide can be flanked at the N-terminus, the C-terminus, or boththe N-terminus and C-terminus by one or more nuclear localizationsignals (NLSs). For example, a Cas9 endonuclease can be flanked by twoNLSs, one NLS located at the N-terminus and the second NLS located atthe C-terminus. The NLS can be any NLS known in the art, such as a SV40NLS. The weight ratio of genome-targeting nucleic acid to site-directedpolypeptide in the RNP can be 1:1. For example, the weight ratio ofsgRNA to Cas9 endonuclease in the RNP can be 1:1.

Target Sequence Selection

Shifts in the location of the 5′ boundary and/or the 3′ boundaryrelative to particular reference loci can be used to facilitate orenhance particular applications of gene editing, which depend in part onthe endonuclease system selected for the editing, as further describedand illustrated herein.

In a first non-limiting example of such target sequence selection, manyendonuclease systems have rules or criteria that can guide the initialselection of potential target sites for cleavage, such as therequirement of a PAM sequence motif in a particular position adjacent tothe DNA cleavage sites in the case of CRISPR Type II or Type Vendonucleases.

In another nonlimiting example of target sequence selection oroptimization, the frequency of off-target activity for a particularcombination of target sequence and gene editing endonuclease can beassessed relative to the frequency of on-target activity. In some cases,cells that have been correctly edited at the desired locus can have aselective advantage relative to other cells. Illustrative, butnonlimiting, examples of a selective advantage include the acquisitionof attributes such as enhanced rates of replication, persistence,resistance to certain conditions, enhanced rates of successfulengraftment or persistence in vivo following introduction into apatient, and other attributes associated with the maintenance orincreased numbers or viability of such cells. In other cases, cells thathave been correctly edited at the desired locus can be positivelyselected for by one or more screening methods used to identify, sort orotherwise select for cells that have been correctly edited. Bothselective advantage and directed selection methods can take advantage ofthe phenotype associated with the correction. In some cases, cells canbe edited two or more times in order to create a second modificationthat creates a new phenotype that is used to select or purify theintended population of cells. Such a second modification could becreated by adding a second gRNA for a selectable or screenable marker.In some cases, cells can be correctly edited at the desired locus usinga DNA fragment that contains the cDNA and also a selectable marker.

Whether any selective advantage is applicable or any directed selectionis to be applied in a particular case, target sequence selection canalso be guided by consideration of off-target frequencies in order toenhance the effectiveness of the application and/or reduce the potentialfor undesired alterations at sites other than the desired target. Asdescribed further and illustrated herein and in the art, the occurrenceof off-target activity can be influenced by a number of factorsincluding similarities and dissimilarities between the target site andvarious off-target sites, as well as the particular endonuclease used.Bioinformatics tools are available that assist in the prediction ofoff-target activity, and frequently such tools can also be used toidentify the most likely sites of off-target activity, which can then beassessed in experimental settings to evaluate relative frequencies ofoff-target to on-target activity, thereby allowing the selection ofsequences that have higher relative on-target activities. Illustrativeexamples of such techniques are provided herein, and others are known inthe art.

gRNAs of the present disclosure can direct editing at a genetic locuswhere editing is desired (e.g., a mutant allele of the USH2A gene). Asused herein, “on-target editing,” “on-target activity,” or “on-targetcleavage” means editing at a genetic locus where editing is desired.

gRNAs disclosed herein can have on-target activity when the gRNA directsediting of the corresponding mutant allele within or near the IVS40mutation.

gRNAs of the present disclosure can also direct editing at a geneticlocus where editing is not desired. As used herein, “off-targetediting,” “off-target activity,” or “off-target cleavage” means editingat a genetic locus where editing is not desired.

Off-target editing can be editing of a wild-type allele of the USH2Agene. Herein, this type of off-target editing is termed “wild-typeoff-target editing,” “wild-type off-target activity,” or “wild-typeoff-target cleavage.” A gRNA disclosed herein can have wild-typeoff-target activity when the gRNA directs editing of a wild-type USH2Aallele.

Off-target editing can be editing of a second gene or locus (e.g.,editing of a genomic sequence that is not a sequence of the USH2A geneor a regulatory sequence of the USH2A gene). Herein, this type ofoff-target editing is termed “genomic off-target editing,” “genomicoff-target activity,” or “genomic off-target cleavage.” gRNAs disclosedherein have genomic off-target activity when the gRNA directs editing ofa genomic sequence that is not a sequence of the USH2A gene or aregulatory sequence of the USH2A gene.

In some examples, wild-type off-target activity of a gRNA can be“minimal.” gRNAs with minimal wild-type off-target activity can bedetermined using methods known in the art, for example, methods based onin silico analysis, in vitro methods, or in vivo methods of determiningthe amount of wild-type off-target editing caused by a gRNA. A gRNA withminimal wild-type off-target activity can cause off-target editing in30% or less of cells, for example, 25% or less of cells, 20% or less ofcells, 15% or less of cells 10% or less of cells, 5% or less of cells,4% or less of cells, 3% or less of cells, 2% or less of cells, 1% orless of cells, 0.5% or less of cells, 0.25% or less of cells, or 0.1% orless of cells. Such determinations can, in some cases, be determinedusing in vitro systems.

In some examples, genomic off-target activity of a gRNA can be“minimal.” gRNAs with minimal genomic off-target activity can bedetermined based on in silico analysis, in vitro methods, or in vivomethods of determining the amount of genomic off-target editing causedby a gRNA. A gRNA with minimal genomic off-target activity can cause atleast one instance of genomic off-target editing in 30% or less of cellssuch as, for example, 25% or less of cells, 20% or less of cells, 15% orless of cells 10% or less of cells, 5% or less of cells, 4% or less ofcells, 3% or less of cells, 2% or less of cells, 1% or less of cells,0.5% or less of cells, 0.25% or less of cells, or 0.1% or less of cells.Such determinations can, in some cases, be determined using in vitrosystems.

Another aspect of target sequence selection relates to homologousrecombination events. Sequences sharing regions of homology can serve asfocal points for homologous recombination events that result in deletionof intervening sequences. Such recombination events occur during thenormal course of replication of chromosomes and other DNA sequences, andalso at other times when DNA sequences are being synthesized, such as inthe case of repairs of double-strand breaks (DSBs), which occur on aregular basis during the normal cell replication cycle but can also beenhanced by the occurrence of various events (such as UV light and otherinducers of DNA breakage) or the presence of certain agents (such asvarious chemical inducers). Many such inducers cause DSBs to occurindiscriminately in the genome, and DSBs can be regularly induced andrepaired in normal cells. During repair, the original sequence can bereconstructed with complete fidelity, however, in some cases, smallinsertions or deletions (referred to as “indels”) are introduced at theDSB site.

DSBs can also be specifically induced at particular locations, as in thecase of the endonucleases systems described herein, which can be used tocause directed or preferential gene modification events at selectedchromosomal locations. The tendency for homologous sequences to besubject to recombination in the context of DNA repair (as well asreplication) can be taken advantage of in a number of circumstances, andis the basis for one application of gene editing systems, such asCRISPR, in which homology directed repair is used to insert a sequenceof interest, provided through use of a “donor” polynucleotide, into adesired chromosomal location.

Regions of homology between particular sequences, which can be smallregions of “microhomology” that can comprise as few as ten base pairs orless, can also be used to bring about desired deletions. For example, asingle DSB can be introduced at a site that exhibits microhomology witha nearby sequence. During the normal course of repair of such DSB, aresult that occurs with high frequency is the deletion of theintervening sequence as a result of recombination being facilitated bythe DSB and concomitant cellular repair process.

In some circumstances, however, selecting target sequences withinregions of homology can also give rise to much larger deletions,including gene fusions (when the deletions are in coding regions), whichmay or may not be desired given the particular circumstances.

The examples provided herein further illustrate the selection of varioustarget regions for the creation of DSBs designed to induce replacementsthat result in restoration of usherin protein function, as well as theselection of specific target sequences within such regions that aredesigned to minimize off-target events relative to on-target events.

Homology Direct Repair (HDR)/Donor Nucleotides

Homology direct repair is a cellular mechanism for repairingdouble-stranded breaks (DSBs). The most common form is homologousrecombination. There are additional pathways for HDR, includingsingle-strand annealing and alternative-HDR. Genome engineering toolsallow researchers to manipulate the cellular homologous recombinationpathways to create site-specific modifications to the genome. It hasbeen found that cells can repair a double-stranded break using asynthetic donor molecule provided in trans. Therefore, by introducing adouble-stranded break near a specific mutation and providing a suitabledonor, targeted changes can be made in the genome. Specific cleavageincreases the rate of HDR more than 1,000 fold above the rate of 1 in10⁶ cells receiving a homologous donor alone. The rate of HDR at aparticular nucleotide is a function of the distance to the cut site, sochoosing overlapping or nearest target sites is important. Gene editingoffers the advantage over gene addition, as correcting in situ leavesthe rest of the genome unperturbed.

Supplied donors for editing by HDR vary markedly but can contain theintended sequence with small or large flanking homology arms to allowannealing to the genomic DNA. The homology regions flanking theintroduced genetic changes can be 30 bp or smaller, or as large as amulti-kilobase cassette that can contain promoters, cDNAs, etc. Bothsingle-stranded and double-stranded oligonucleotide donors have beenused. These oligonucleotides range in size from less than 100 nt to overmany kb, though longer ssDNA can also be generated and used.Double-stranded donors can be used, including PCR amplicons, plasmids,and mini-circles. In general, it has been found that an AAV vector canbe a very effective means of delivery of a donor template, though thepackaging limits for individual donors is <5 kb. Active transcription ofthe donor increased HDR three-fold, indicating the inclusion of promotermay increase conversion. Conversely, CpG methylation of the donordecreased gene expression and HDR.

Donor nucleotides for correcting mutations often are small (<300 bp).This is advantageous, as HDR efficiencies may be inversely related tothe size of the donor molecule. Also, it is expected that the donortemplates can fit into size constrained AAV molecules, which have beenshown to be an effective means of donor template delivery.

In addition to wildtype endonucleases, such as Cas9, nickase variantsexist that have one or the other nuclease domain inactivated resultingin cutting of only one DNA strand. HDR can be directed from individualCas nickases or using pairs of nickases that flank the target area.Donors can be single-stranded, nicked, or dsDNA.

The donor DNA can be supplied with the nuclease or independently by avariety of different methods, for example by transfection, nanoparticle,microinjection, or viral transduction. A range of tethering options havebeen proposed to increase the availability of the donors for HDR.Examples include attaching the donor to the nuclease, attaching to DNAbinding proteins that bind nearby, or attaching to proteins that areinvolved in DNA end binding or repair.

The repair pathway choice can be guided by a number of cultureconditions, such as those that influence cell cycling, or by targetingof DNA repair and associated proteins. For example, to increase HDR, keyNHEJ molecules can be suppressed, such as KU70, KU80 or DNA ligase IV.

Without a donor present, the ends from a DNA break or ends fromdifferent breaks can be joined using the several non-homologous repairpathways in which the DNA ends are joined with little or no base-pairingat the junction. In addition to canonical NHEJ, there are similar repairmechanisms, such as ANHEJ. If there are two breaks, the interveningsegment can be deleted or inverted. NHEJ repair pathways can lead toinsertions, deletions or mutations at the joints.

NHEJ was used to insert a 15-kb inducible gene expression cassette intoa defined locus in human cell lines after nuclease cleavage. The methodsof insertion of large inducible gene expression cassettes have beendescribed [Maresca, M., Lin, V. G., Guo, N. & Yang, Y., Genome Res 23,539-546 (2013), Suzuki et al. Nature, 540, 144-149 (2016)].

In addition to genome editing by NHEJ or HDR, site-specific geneinsertions have been conducted that use both the NHEJ pathway and HDR. Acombination approach can be applicable in certain settings, possiblyincluding intron/exon borders. NHEJ may prove effective for ligation inthe intron, while the error-free HDR may be better suited in the codingregion.

Illustrative modifications within the USH2A gene include replacementswithin or near (proximal) to the mutations referred to above, such aswithin the region of less than 3 kb, less than 2 kb, less than 1 kb,less than 0.5 kb upstream or downstream of the specific mutation. Giventhe relatively wide variations of mutations in the USH2A gene, it willbe appreciated that numerous variations of the replacements referencedabove (including without limitation larger as well as smallerdeletions), would be expected to result in restoration of the usherinprotein function.

Such variants can include replacements that are larger in the 5′ and/or3′ direction than the specific mutation in question, or smaller ineither direction. Accordingly, by “near” or “proximal” with respect tospecific replacements, it is intended that the SSB or DSB locusassociated with a desired replacement boundary (also referred to hereinas an endpoint) can be within a region that is less than about 3 kb fromthe reference locus. e.g., the mutation site. The SSB or DSB locus canbe more proximal and within 2 kb, within 1 kb, within 0.5 kb, or within0.1 kb. In the case of a small replacement, the desired endpoint can beat or “adjacent to” the reference locus, by which it is intended thatthe endpoint can be within 100 bp, within 50 bp, within 25 bp, or lessthan about 10 bp to 5 bp from the reference locus.

Larger or smaller replacements can provide the same benefit, as long asthe usherin protein function is restored. It is thus expected that manyvariations of the replacements described and illustrated herein can beeffective for ameliorating Usher Syndrome Type 2A.

The terms “near” or “proximal” with respect to the SSBs or DSBs refer tothe SSBs or DSBs being within 2 kb, within 1 kb, within 0.5 kb, within0.25 kb, within 0.2 kb, within 0.1 kb, within 50 bp, within 25 bp,within 20 bp, within 15 bp, within 10 bp, within 5 bp of the IVS40mutation. The SSB or DSB locus can also be within 2 kb, within 1 kb,within 0.5 kb, within 0.25 kb, within 0.2 kb, or within 0.1 kb, within50 bp, within 25 bp, within 20 bp, within 15 bp, within 10 bp, within 5bp of intron 40.

Nucleic Acid Modifications (Chemical and Structural Modifications)

In some cases, polynucleotides introduced into cells can comprise one ormore modifications that can be used individually or in combination, forexample, to enhance activity, stability or specificity, alter delivery,reduce innate immune responses in host cells, or for other enhancements,as further described herein and known in the art.

In certain examples, modified polynucleotides can be used in theCRISPR/Cas9/Cpf1 system, in which case the guide RNAs (eithersingle-molecule guides or double-molecule guides) and/or a DNA or an RNAencoding a Cas or Cpf1 endonuclease introduced into a cell can bemodified, as described and illustrated below. Such modifiedpolynucleotides can be used in the CRISPR/Cas9/Cpf1 system to edit anyone or more genomic loci.

Using the CRISPR/Cas9/Cpf1 system for purposes of non-limitingillustrations of such uses, modifications of guide RNAs can be used toenhance the formation or stability of the CRISPR/Cas9/Cpf1 genomeediting complex comprising guide RNAs, which can be single-moleculeguides or double-molecule, and a Cas or Cpf1 endonuclease. Modificationsof guide RNAs can also or alternatively be used to enhance theinitiation, stability or kinetics of interactions between the genomeediting complex with the target sequence in the genome, which can beused, for example, to enhance on-target activity. Modifications of guideRNAs can also or alternatively be used to enhance specificity, e.g., therelative rates of genome editing at the on-target site as compared toeffects at other (off-target) sites.

Modifications can also or alternatively be used to increase thestability of a guide RNA, e.g., by increasing its resistance todegradation by ribonucleases (RNases) present in a cell, thereby causingits half-life in the cell to be increased. Modifications enhancing guideRNA half-life can be particularly useful in aspects in which a Cas orCpf1 endonuclease is introduced into the cell to be edited via an RNAthat needs to be translated in order to generate endonuclease, becauseincreasing the half-life of guide RNAs introduced at the same time asthe RNA encoding the endonuclease can be used to increase the time thatthe guide RNAs and the encoded Cas or Cpf1 endonuclease co-exist in thecell.

Modifications can also or alternatively be used to decrease thelikelihood or degree to which RNAs introduced into cells elicit innateimmune responses. Such responses, which have been well characterized inthe context of RNA interference (RNAi), including small-interfering RNAs(siRNAs), as described below and in the art, tend to be associated withreduced half-life of the RNA and/or the elicitation of cytokines orother factors associated with immune responses.

One or more types of modifications can also be made to RNAs encoding anendonuclease that are introduced into a cell, including, withoutlimitation, modifications that enhance the stability of the RNA (such asby increasing its degradation by RNAses present in the cell),modifications that enhance translation of the resulting product (i.e.the endonuclease), and/or modifications that decrease the likelihood ordegree to which the RNAs introduced into cells elicit innate immuneresponses.

Combinations of modifications, such as the foregoing and others, canlikewise be used. In the case of CRISPR/Cas9/Cpf1, for example, one ormore types of modifications can be made to guide RNAs (including thoseexemplified above), and/or one or more types of modifications can bemade to RNAs encoding Cas endonuclease (including those exemplifiedabove).

By way of illustration, guide RNAs used in the CRISPR/Cas9/Cpf1 system,or other smaller RNAs can be readily synthesized by chemical means,enabling a number of modifications to be readily incorporated, asillustrated below and described in the art. While chemical syntheticprocedures are continually expanding, purifications of such RNAs byprocedures such as high-performance liquid chromatography (HPLC, whichavoids the use of gels such as PAGE) tends to become more challenging aspolynucleotide lengths increase significantly beyond a hundred or sonucleotides. One approach that can be used for generating chemicallymodified RNAs of greater length is to produce two or more molecules thatare ligated together. Much longer RNAs, such as those encoding a Cas9endonuclease, are more readily generated enzymatically. While fewertypes of modifications are available for use in enzymatically producedRNAs, there are still modifications that can be used to, e.g., enhancestability, reduce the likelihood or degree of innate immune response,and/or enhance other attributes, as described further below and in theart; and new types of modifications are regularly being developed.

By way of illustration of various types of modifications, especiallythose used frequently with smaller chemically synthesized RNAs,modifications can comprise one or more nucleotides modified at the 2′position of the sugar, in some aspects a 2′-O-alkyl, 2′-O-alkyl-O-alkyl,or 2′-fluoro-modified nucleotide. In some examples, RNA modificationscan comprise 2′-fluoro, 2′-amino or 2′-O-methyl modifications on theribose of pyrimidines, abasic residues, or an inverted base at the 3′end of the RNA. Such modifications can be routinely incorporated intooligonucleotides and these oligonucleotides have been shown to have ahigher Tm (i.e., higher target binding affinity) than2′-deoxyoligonucleotides against a given target.

A number of nucleotide and nucleoside modifications have been shown tomake the oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligonucleotide; these modifiedoligos survive intact for a longer time than unmodifiedoligonucleotides. Specific examples of modified oligonucleotides includethose comprising modified backbones, for example, phosphorothioates,phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. Some oligonucleotides are oligonucleotides withphosphorothioate backbones and those with heteroatom backbones,particularly CH₂—NH—O—CH₂, CH, ˜N(CH₃)˜O˜CH₂ (known as amethylene(methylimino) or MMI backbone), CH₂—O—N (CH₃)—CH₂,CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)— CH₂—CH₂ backbones, wherein thenative phosphodiester backbone is represented as O— P— O— CH); amidebackbones [see De Mesmaeker et al., Ace. Chem. Res., 28:366-374 (1995)];morpholino backbone structures (see Summerton and Weller, U.S. Pat. No.5,034,506); peptide nucleic acid (PNA) backbone (wherein thephosphodiester backbone of the oligonucleotide is replaced with apolyamide backbone, the nucleotides being bound directly or indirectlyto the aza nitrogen atoms of the polyamide backbone, see Nielsen et al.,Science 1991, 254, 1497). Phosphorus-containing linkages include, butare not limited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3′alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds are described in Braasch andCorey, Biochemistry, 41(14): 4503-4510 (2002); Genesis, Volume 30, Issue3, (2001); Heasman, Dev. Biol., 243: 209-214 (2002); Nasevicius et al.,Nat. Genet., 26:216-220 (2000); Lacerra et al., Proc. Natl. Acad. Sci.,97: 9591-9596 (2000); and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wanget al., J. Am. Chem. Soc., 122: 8595-8602 (2000).

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S, and CH₂ component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439.

One or more substituted sugar moieties can also be included, e.g., oneof the following at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃, OCH₃O(CH₂)n CH₃, O(CH₂)n NH₂, or O(CH₂)n CH₃, where n is from 1 to about 10;C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl oraralkyl; Cl; Br; CN; CF₃; OCF₃; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; SOCH₃; SO₂ CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. In some aspects, amodification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl)) (Martin et al, HeIv. Chim. Acta, 1995, 78, 486).Other modifications include 2′-methoxy (2′-O—CH₃), 2′-propoxy (2′-OCH₂CH₂CH₃) and 2′-fluoro (2′-F). Similar modifications can also be made atother positions on the oligonucleotide, particularly the 3′ position ofthe sugar on the 3′ terminal nucleotide and the 5′ position of 5′terminal nucleotide. Oligonucleotides can also have sugar mimetics, suchas cyclobutyls in place of the pentofuranosyl group.

In some examples, both a sugar and an internucleoside linkage, i.e., thebackbone, of the nucleotide units can be replaced with novel groups. Thebase units can be maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, anoligonucleotide mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide can bereplaced with an amide containing backbone, for example, anaminoethylglycine backbone. The nucleobases can be retained and bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative U.S. patents that teach the preparation of PNAcompounds comprise, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262. Further teaching of PNA compounds can be foundin Nielsen et al, Science, 254: 1497-1500 (1991).

Guide RNAs can also include, additionally or alternatively, nucleobase(often referred to in the art simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude adenine (A), guanine (G), thymine (T), cytosine (C), and uracil(U). Modified nucleobases include nucleobases found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2′ deoxycytosine and often referred to in theart as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC andgentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine,2-(methylamino) adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil,2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine,7-deazaguanine, N6 (6-aminohexyl) adenine, and 2,6-diaminopurine.Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, pp.75-77 (1980); Gebeyehu et al., Nucl. Acids Res. 15:4513 (1997). A“universal” base known in the art, e.g., inosine, can also be included.5-Me-C substitutions have been shown to increase nucleic acid duplexstability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu,B., eds., Antisense Research and Applications, CRC Press, Boca Raton,1993, pp. 276-278) and are aspects of base substitutions.

Modified nucleobases can comprise other synthetic and naturalnucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and3-deazaadenine.

Further, nucleobases can comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopedia of PolymerScience and Engineering’, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandleChemie, International Edition', 1991, 30, page 613, and those disclosedby Sanghvi, Y. S., Chapter 15, Antisense Research and Applications',pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993.Certain of these nucleobases are particularly useful for increasing thebinding affinity of the oligomeric compounds of the invention. Theseinclude 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, comprising 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds, ‘Antisense Research andApplications’, CRC Press, Boca Raton, 1993, pp. 276-278) and are aspectsof base substitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications. Modified nucleobases aredescribed in U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;5,587,469; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 5,763,588;5,830,653; 6,005,096; and U.S. Patent Application Publication2003/0158403.

Thus, the term “modified” refers to a non-natural sugar, phosphate, orbase that is incorporated into a guide RNA, an endonuclease, or both aguide RNA and an endonuclease. It is not necessary for all positions ina given oligonucleotide to be uniformly modified, and in fact more thanone of the aforementioned modifications can be incorporated in a singleoligonucleotide, or even in a single nucleoside within anoligonucleotide.

The guide RNAs and/or mRNA (or DNA) encoding an endonuclease can bechemically linked to one or more moieties or conjugates that enhance theactivity, cellular distribution, or cellular uptake of theoligonucleotide. Such moieties comprise, but are not limited to, lipidmoieties such as a cholesterol moiety [Letsinger et al., Proc. Natl.Acad. Sci. USA, 86: 6553-6556 (1989)]; cholic acid [Manoharan et al.,Bioorg. Med. Chem. Let., 4: 1053-1060 (1994)]; a thioether, e.g.,hexyl-S-tritylthiol [Manoharan et al, Ann. N. Y. Acad. Sci., 660:306-309 (1992) and Manoharan et al., Bioorg. Med. Chem. Let., 3:2765-2770 (1993)]; a thiocholesterol [Oberhauser et al., Nucl. AcidsRes., 20: 533-538 (1992)]; an aliphatic chain, e.g., dodecandiol orundecyl residues [Kabanov et al., FEBS Lett., 259: 327-330 (1990) andSvinarchuk et al., Biochimie, 75: 49-54 (1993)]; a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate [Manoharan et al.,Tetrahedron Lett., 36: 3651-3654 (1995) and Shea et al., Nucl. AcidsRes., 18: 3777-3783 (1990)]; a polyamine or a polyethylene glycol chain[Mancharan et al., Nucleosides & Nucleotides, 14: 969-973 (1995)];adamantane acetic acid [Manoharan et al., Tetrahedron Lett., 36:3651-3654 (1995)]; a palmityl moiety [(Mishra et al., Biochim. Biophys.Acta, 1264: 229-237 (1995)]; or an octadecylamine orhexylamino-carbonyl-t oxycholesterol moiety [Crooke et al., J.Pharmacol. Exp. Ther., 277: 923-937 (1996)]. See also U.S. Pat. Nos.4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599, 928 and 5,688,941.

Sugars and other moieties can be used to target proteins and complexescomprising nucleotides, such as cationic polysomes and liposomes, toparticular sites. For example, hepatic cell directed transfer can bemediated via asialoglycoprotein receptors (ASGPRs); see, e.g., Hu, etal., Protein Pept Lett. 21(10):1025-30 (2014). Other systems known inthe art and regularly developed can be used to target biomolecules ofuse in the present case and/or complexes thereof to particular targetcells of interest.

These targeting moieties or conjugates can include conjugate groupscovalently bound to functional groups, such as primary or secondaryhydroxyl groups. Conjugate groups of the present disclosure includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of this presentdisclosure, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentdisclosure. Representative conjugate groups are disclosed inInternational Patent Application No. PCT/US92/09196, filed Oct. 23, 1992(published as WO1993007883), and U.S. Pat. No. 6,287,860. Conjugatemoieties include, but are not limited to, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, apolyamine or a polyethylene glycol chain, or adamantane acetic acid, apalmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941.

Longer polynucleotides that are less amenable to chemical synthesis andare typically produced by enzymatic synthesis can also be modified byvarious means. Such modifications can include, for example, theintroduction of certain nucleotide analogs, the incorporation ofparticular sequences or other moieties at the 5′ or 3′ ends ofmolecules, and other modifications. By way of illustration, the mRNAencoding Cas9 is approximately 4 kb in length and can be synthesized byin vitro transcription. Modifications to the mRNA can be applied to,e.g., increase its translation or stability (such as by increasing itsresistance to degradation with a cell), or to reduce the tendency of theRNA to elicit an innate immune response that is often observed in cellsfollowing introduction of exogenous RNAs, particularly longer RNAs suchas that encoding Cas9.

Numerous such modifications have been described in the art, such aspolyA tails, 5′ cap analogs (e.g., Anti Reverse Cap Analog (ARCA) orm7G(5′)ppp(5′)G (mCAP)), modified 5′ or 3′ untranslated regions (UTRs),use of modified bases (such as Pseudo-UTP, 2-Thio-UTP,5-Methylcytidine-5′-Triphosphate (5-Methyl-CTP) or N6-Methyl-ATP), ortreatment with phosphatase to remove 5′ terminal phosphates. These andother modifications are known in the art, and new modifications of RNAsare regularly being developed.

There are numerous commercial suppliers of modified RNAs, including forexample, TriLink Biotech, AxoLabs, Bio-Synthesis Inc., Dharmacon andmany others. As described by TriLink, for example, 5-Methyl-CTP can beused to impart desirable characteristics, such as increased nucleasestability, increased translation or reduced interaction of innate immunereceptors with in vitro transcribed RNA.5-Methylcytidine-5′-Triphosphate (5-Methyl-CTP), N6-Methyl-ATP, as wellas Pseudo-UTP and 2-Thio-UTP, have also been shown to reduce innateimmune stimulation in culture and in vivo while enhancing translation,as illustrated in publications by Kormann et al. and Warren et al.referred to below.

It has been shown that chemically modified mRNA delivered in vivo can beused to achieve improved therapeutic effects; see, e.g., Kormann et al.,Nature Biotechnology 29, 154-157 (2011). Such modifications can be used,for example, to increase the stability of the RNA molecule and/or reduceits immunogenicity. Using chemical modifications such as Pseudo-U,N6-Methyl-A, 2-Thio-U and 5-Methyl-C, it was found that substitutingjust one quarter of the uridine and cytidine residues with 2-Thio-U and5-Methyl-C respectively resulted in a significant decrease in toll-likereceptor (TLR) mediated recognition of the mRNA in mice. By reducing theactivation of the innate immune system, these modifications can be usedto effectively increase the stability and longevity of the mRNA in vivo;see, e.g., Kormann et al., supra.

It has also been shown that repeated administration of syntheticmessenger RNAs incorporating modifications designed to bypass innateanti-viral responses can reprogram differentiated human cells topluripotency. See, e.g., Warren, et al., Cell Stem Cell, 7(5):618-30(2010). Such modified mRNAs that act as primary reprogramming proteinscan be an efficient means of reprogramming multiple human cell types.Such cells are referred to as induced pluripotency stem cells (iPSCs),and it was found that enzymatically synthesized RNA incorporating5-Methyl-CTP, Pseudo-UTP and an Anti Reverse Cap Analog (ARCA) could beused to effectively evade the cell's antiviral response; see, e.g.,Warren et al., supra.

Other modifications of polynucleotides described in the art include, forexample, the use of polyA tails, the addition of 5′ cap analogs such as(m7G(5′)ppp(5′)G (mCAP)), modifications of 5′ or 3′ untranslated regions(UTRs), or treatment with phosphatase to remove 5′ terminalphosphates—and new approaches are regularly being developed.

A number of compositions and techniques applicable to the generation ofmodified RNAs for use herein have been developed in connection with themodification of RNA interference (RNAi), including small-interferingRNAs (siRNAs). siRNAs present particular challenges in vivo becausetheir effects on gene silencing via mRNA interference are generallytransient, which can require repeat administration. In addition, siRNAsare double-stranded RNAs (dsRNA) and mammalian cells have immuneresponses that have evolved to detect and neutralize dsRNA, which isoften a by-product of viral infection. Thus, there are mammalian enzymessuch as PKR (dsRNA-responsive kinase), and potentially retinoicacid-inducible gene I (RIG-I), that can mediate cellular responses todsRNA, as well as Toll-like receptors (such as TLR3, TLR7 and TLR8) thatcan trigger the induction of cytokines in response to such molecules;see, e.g., the reviews by Angart et al., Pharmaceuticals (Basel) 6(4):440-468 (2013); Kanasty et al., Molecular Therapy 20(3): 513-524 (2012);Burnett et al., Biotechnol J. 6(9):1130-46 (2011); Judge and MacLachlan,Hum Gene Ther 19(2):111-24 (2008).

A large variety of modifications have been developed and applied toenhance RNA stability, reduce innate immune responses, and/or achieveother benefits that can be useful in connection with the introduction ofpolynucleotides into human cells, as described herein; see, e.g., thereviews by Whitehead K A et al., Annual Review of Chemical andBiomolecular Engineering, 2: 77-96 (2011); Gaglione and Messere, MiniRev Med Chem, 10(7):578-95 (2010); Chernolovskaya et al, Curr Opin MolTher., 12(2):158-67 (2010); Deleavey et al., Curr Protoc Nucleic AcidChem Chapter 16:Unit 16.3 (2009); Behlke, Oligonucleotides 18(4):305-19(2008); Fucini et al., Nucleic Acid Ther 22(3): 205-210 (2012); Bremsenet al., Front Genet 3:154 (2012).

As noted above, there are a number of commercial suppliers of modifiedRNAs, many of which have specialized in modifications designed toimprove the effectiveness of siRNAs. A variety of approaches are offeredbased on various findings reported in the literature. For example,Dharmacon notes that replacement of a non-bridging oxygen with sulfur(phosphorothioate, PS) has been extensively used to improve nucleaseresistance of siRNAs, as reported by Kole, Nature Reviews Drug Discovery11:125-140 (2012). Modifications of the 2′-position of the ribose havebeen reported to improve nuclease resistance of the internucleotidephosphate bond while increasing duplex stability (Tm), which has alsobeen shown to provide protection from immune activation. A combinationof moderate PS backbone modifications with small, well-tolerated2′-substitutions (2′-O-Methyl, 2′-Fluoro, 2′-Hydro) have been associatedwith highly stable siRNAs for applications in vivo, as reported bySoutschek et al. Nature 432:173-178 (2004); and 2′-O-Methylmodifications have been reported to be effective in improving stabilityas reported by Volkov, Oligonucleotides 19:191-202 (2009). With respectto decreasing the induction of innate immune responses, modifyingspecific sequences with 2′-O-Methyl, 2′-Fluoro, 2′-Hydro have beenreported to reduce TLR7/TLR8 interaction while generally preservingsilencing activity; see, e.g., Judge et al., Mol. Ther. 13:494-505(2006); and Cekaite et al., J. Mol. Biol. 365:90-108 (2007). Additionalmodifications, such as 2-thiouracil, pseudouracil, 5-methylcytosine,5-methyluracil, and N6-methyladenosine have also been shown to minimizethe immune effects mediated by TLR3, TLR7, and TLR8; see, e.g., Kariko,K. et al., Immunity 23:165-175 (2005).

As is also known in the art, and commercially available, a number ofconjugates can be applied to polynucleotides, such as RNAs, for useherein that can enhance their delivery and/or uptake by cells, includingfor example, cholesterol, tocopherol and folic acid, lipids, peptides,polymers, linkers and aptamers; see, e.g., the review by Winkler, Ther.Deliv. 4:791-809 (2013).

Codon-Optimization

A polynucleotide encoding a site-directed polypeptide can becodon-optimized according to methods standard in the art for expressionin the cell containing the target DNA of interest. For example, if theintended target nucleic acid is in a human cell, a human codon-optimizedpolynucleotide encoding Cas9 is contemplated for use for producing theCas9 polypeptide.

Nucleic Acids Encoding System Components

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a genome-targeting nucleic acid of the disclosure, asite-directed polypeptide of the disclosure, and/or any nucleic acid orproteinaceous molecule necessary to carry out the aspects of the methodsof the disclosure.

The nucleic acid encoding a genome-targeting nucleic acid of thedisclosure, a site-directed polypeptide of the disclosure, and/or anynucleic acid or proteinaceous molecule necessary to carry out theaspects of the methods of the disclosure can comprise a vector (e.g., arecombinant expression vector).

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double-stranded DNAloop into which additional nucleic acid segments can be ligated. Anothertype of vector is a viral vector; wherein additional nucleic acidsegments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome.

In some examples, vectors can be capable of directing the expression ofnucleic acids to which they are operatively linked. Such vectors arereferred to herein as “recombinant expression vectors”, or more simply“expression vectors”, which serve equivalent functions.

The term “operably linked” means that the nucleotide sequence ofinterest is linked to regulatory sequence(s) in a manner that allows forexpression of the nucleotide sequence. The term “regulatory sequence” isintended to include, for example, promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those that directconstitutive expression of a nucleotide sequence in many types of hostcells, and those that direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Itwill be appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the targetcell, the level of expression desired, and the like.

Expression vectors contemplated include, but are not limited to, viralvectors based on vaccinia virus, poliovirus, adenovirus,adeno-associated virus, SV40, herpes simplex virus, humanimmunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleennecrosis virus, and vectors derived from retroviruses such as RousSarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus,human immunodeficiency virus, myeloproliferative sarcoma virus, andmammary tumor virus) and other recombinant vectors. Other vectorscontemplated for eukaryotic target cells include, but are not limitedto, the vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).Additional vectors contemplated for eukaryotic target cells include, butare not limited to, the vectors pCTx-1, pCTx-2, and pCTx-3. Othervectors can be used so long as they are compatible with the host cell.

In some examples, a vector can comprise one or more transcription and/ortranslation control elements. Depending on the host/vector systemutilized, any of a number of suitable transcription and translationcontrol elements, including constitutive and inducible promoters,transcription enhancer elements, transcription terminators, etc. can beused in the expression vector. The vector can be a self-inactivatingvector that either inactivates the viral sequences or the components ofthe CRISPR machinery or other elements.

Non-limiting examples of suitable eukaryotic promoters (i.e., promotersfunctional in a eukaryotic cell) include those from cytomegalovirus(CMV) immediate early, herpes simplex virus (HSV) thymidine kinase,early and late SV40, long terminal repeats (LTRs) from retrovirus, humanelongation factor-1 promoter (EF1), a hybrid construct comprising thecytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter(CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1locus promoter (PGK), and mouse metallothionein-I.

For expressing small RNAs, including guide RNAs used in connection withCas endonuclease, various promoters such as RNA polymerase IIIpromoters, including for example U6 and H1, can be advantageous.Descriptions of and parameters for enhancing the use of such promotersare known in art, and additional information and approaches areregularly being described; see, e.g., Ma, H. et al., MolecularTherapy—Nucleic Acids 3, e161 (2014) doi:10.1038/mtna.2014.12.

The expression vector can also contain a ribosome binding site fortranslation initiation and a transcription terminator. The expressionvector can also comprise appropriate sequences for amplifyingexpression. The expression vector can also include nucleotide sequencesencoding non-native tags (e.g., histidine tag, hemagglutinin tag, greenfluorescent protein, etc.) that are fused to the site-directedpolypeptide, thus resulting in a fusion protein.

A promoter can be an inducible promoter (e.g., a heat shock promoter,tetracycline-regulated promoter, steroid-regulated promoter,metal-regulated promoter, estrogen receptor-regulated promoter, etc.).The promoter can be a constitutive promoter (e.g., CMV promoter, UBCpromoter). In some cases, the promoter can be a spatially restrictedand/or temporally restricted promoter (e.g., a tissue specific promoter,a cell type specific promoter, etc.).

The nucleic acid encoding a genome-targeting nucleic acid of thedisclosure and/or a site-directed polypeptide can be packaged into or onthe surface of delivery vehicles for delivery to cells. Deliveryvehicles contemplated include, but are not limited to, nanospheres,liposomes, quantum dots, nanoparticles, polyethylene glycol particles,hydrogels, and micelles. As described in the art, a variety of targetingmoieties can be used to enhance the preferential interaction of suchvehicles with desired cell types or locations.

Introduction of the complexes, polypeptides, and nucleic acids of thedisclosure into cells can occur by viral or bacteriophage infection,transfection, conjugation, protoplast fusion, lipofection,electroporation, nucleofection, calcium phosphate precipitation,polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediatedtransfection, liposome-mediated transfection, particle gun technology,calcium phosphate precipitation, direct micro-injection,nanoparticle-mediated nucleic acid delivery, and the like.

microRNA (miRNA)

Another class of gene regulatory regions is microRNA (miRNA) bindingsites. miRNAs are non-coding RNAs that play key roles inpost-transcriptional gene regulation. miRNAs reportedly regulate theexpression of a large number of mammalian protein-encoding genes.Specific and potent gene silencing by double-stranded RNA (RNAi) wasdiscovered, plus additional small noncoding RNA (Canver, M. C. et al.,Nature (2015)). The largest class of non-coding RNAs important for genesilencing is miRNAs. In mammals, miRNAs are first transcribed as longRNA transcripts, which can be separate transcriptional units, part ofprotein introns, or other transcripts. The long transcripts are calledprimary miRNA (pri-miRNA) that include imperfectly base-paired hairpinstructures. These pri-miRNA can be cleaved into one or more shorterprecursor miRNAs (pre-miRNAs) by Microprocessor, a protein complex inthe nucleus, involving Drosha.

Pre-miRNAs are short stem loops ˜70 nucleotides in length with a2-nucleotide 3′-overhang that are exported into the mature 19-25nucleotide miRNA:miRNA* duplexes. The miRNA strand with lower basepairing stability (the guide strand) can be loaded onto the RNA-inducedsilencing complex (RISC). The passenger guide strand (marked with *),can be functional, but is usually degraded. The mature miRNA tethersRISC to partly complementary sequence motifs in target mRNAspredominantly found within the 3′ untranslated regions (UTRs) andinduces posttranscriptional gene silencing (Bartel, D. P. Cell 136,215-233 (2009); Saj, A. & Lai, E. C. Curr Opin Genet Dev 21, 504-510(2011)).

miRNAs can be important in development, differentiation, cell cycle andgrowth control, and in virtually all biological pathways in mammals andother multicellular organisms. miRNAs can also be involved in cell cyclecontrol, apoptosis and stem cell differentiation, hematopoiesis,hypoxia, muscle development, neurogenesis, insulin secretion,cholesterol metabolism, aging, viral replication and immune responses.

A single miRNA can target hundreds of different mRNA transcripts, whilean individual transcript can be targeted by many different miRNAs. Morethan 28645 microRNAs have been annotated in the latest release ofmiRBase (v.21). Some miRNAs can be encoded by multiple loci, some ofwhich can be expressed from tandemly co-transcribed clusters. Thefeatures allow for complex regulatory networks with multiple pathwaysand feedback controls. miRNAs can be integral parts of these feedbackand regulatory circuits and can help regulate gene expression by keepingprotein production within limits (Herranz, H. & Cohen, S. M. Genes Dev24, 1339-1344 (2010); Posadas, D. M. & Carthew, R. W. Curr Opin GenetDev 27, 1-6 (2014)).

miRNA can also be important in a large number of human diseases that areassociated with abnormal miRNA expression. This association underscoresthe importance of the miRNA regulatory pathway. Recent miRNA deletionstudies have linked miRNA with regulation of the immune responses(Stern-Ginossar, N. et al., Science 317, 376-381 (2007)).

miRNA also have a strong link to cancer and can play a role in differenttypes of cancer. miRNAs have been found to be downregulated in a numberof tumors. miRNA can be important in the regulation of keycancer-related pathways, such as cell cycle control and the DNA damageresponse, and can therefore be used in diagnosis and can be targetedclinically. miRNAs can delicately regulate the balance of angiogenesis,such that experiments depleting all miRNAs suppress tumor angiogenesis(Chen, S. et al., Genes Dev 28, 1054-1067 (2014)).

As has been shown for protein coding genes, miRNA genes can also besubject to epigenetic changes occurring with cancer. Many miRNA loci canbe associated with CpG islands increasing their opportunity forregulation by DNA methylation (Weber, B., Stresemann, C., Brueckner, B.& Lyko, F. Cell Cycle 6, 1001-1005 (2007)). The majority of studies haveused treatment with chromatin remodeling drugs to reveal epigeneticallysilenced miRNAs.

In addition to their role in RNA silencing, miRNAs can also activatetranslation (Posadas, D. M. & Carthew, R. W. Curr Opin Genet Dev 27, 1-6(2014)). Knocking out these sites can lead to decreased expression ofthe targeted gene, while introducing these sites can increaseexpression.

Individual miRNAs can be knocked out most effectively by mutating theseed sequence (bases 2-8 of the microRNA), which can be important forbinding specificity.

Cleavage in this region, followed by mis-repair by NHEJ can effectivelyabolish miRNA function by blocking binding to target sites. miRNAs couldalso be inhibited by specific targeting of the special loop regionadjacent to the palindromic sequence. Catalytically inactive Cas9 canalso be used to inhibit shRNA expression (Zhao, Y. et al., Sci Rep 4,3943 (2014)). In addition to targeting the miRNA, the binding sites canalso be targeted and mutated to prevent the silencing by miRNA.

According to the present disclosure, any of the miRNAs or their bindingsites can be incorporated into the compositions of the invention.

The compositions can have a region such as, but not limited to, a regioncomprising the sequence of any of the miRNAs listed in SEQ ID NOs:613-4696, the reverse complement of the miRNAs listed in SEQ ID NOs:613-4696, or the miRNA anti-seed region of any of the miRNAs listed inSEQ ID NOs: 613-4696.

The compositions of the invention can comprise one or more miRNA targetsequences, miRNA sequences, or miRNA seeds. Such sequences cancorrespond to any known miRNA such as those taught in US Publication No.2005/0261218 and US Publication No. 2005/0059005. As a non-limitingexample, known miRNAs, their sequences, and their binding site sequencesin the human genome are listed in SEQ ID NOs: 613-4696.

A miRNA sequence comprises a “seed” region, i.e., a sequence in theregion of positions 2-8 of the mature miRNA, which sequence has perfectWatson-Crick complementarity to the miRNA target sequence. A miRNA seedcan comprise positions 2-8 or 2-7 of the mature microRNA. In someexamples, a miRNA seed can comprise 7 nucleotides (e.g., nucleotides 2-8of the mature microRNA), wherein the seed-complementary site in thecorresponding miRNA target is flanked by an adenine (A) opposed to miRNAposition 1. In some examples, a miRNA seed can comprise 6 nucleotides(e.g., nucleotides 2-7 of the mature microRNA), wherein theseed-complementary site in the corresponding miRNA target is flanked byan adenine (A) opposed to microRNA position 1. See for example, GrimsonA, Farh K K, Johnston W K, Garrett-Engele P, Lim L P, Bartel D P; MolCell. 2007 Jul. 6; 27(1):91-105. The bases of the miRNA seed havecomplete complementarity with the target sequence.

Identification of miRNA, miRNA target regions, and their expressionpatterns and role in biology have been reported (Bonauer et al., CurrDrug Targets 2010 11:943-949; Anand and Cheresh Curr Opin Hematol 201118:171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec. 20.doi: 10.1038/leu.2011.356); Bartel Cell 2009 136:215-233; Landgraf etal, Cell, 2007 129:1401-1414; Gentner and Naldini, Tissue Antigens. 201280:393-403.

For example, if the composition is not intended to be delivered to theliver but ends up there, then miR-122, a miRNA abundant in liver, caninhibit the expression of the sequence delivered if one or multipletarget sites of miR-122 are engineered into the polynucleotide encodingthat target sequence. Introduction of one or multiple binding sites fordifferent miRNA can be engineered to further decrease the longevity,stability, and protein translation hence providing an additional layerof tenability.

As used herein, the term “miRNA site” refers to a miRNA target site or amiRNA recognition site, or any nucleotide sequence to which a miRNAbinds or associates. It should be understood that “binding” can followtraditional Watson-Crick hybridization rules or can reflect any stableassociation of the miRNA with the target sequence at or adjacent to themiRNA site.

Conversely, for the purposes of the compositions of the presentdisclosure, miRNA binding sites can be engineered out of (i.e., removedfrom) sequences in which they naturally occur in order to increaseprotein expression in specific tissues. For example, miR-122 bindingsites can be removed to improve protein expression in the liver.

Specifically, miRNAs are known to be differentially expressed in immunecells (also called hematopoietic cells), such as antigen presentingcells (APCs) (e.g., dendritic cells and macrophages), macrophages,monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killercells, etc. Immune cell specific miRNAs are involved in immunogenicity,autoimmunity, the immune-response to infection, inflammation, as well asunwanted immune response after gene therapy and tissue/organtransplantation. Immune cells specific microRNAs also regulate manyaspects of development, proliferation, differentiation and apoptosis ofhematopoietic cells (immune cells). For example, miR-142 and miR-146 areexclusively expressed in the immune cells, particularly abundant inmyeloid dendritic cells. Introducing the miR-142 binding site into the3′-UTR of a polypeptide of the present disclosure can selectivelysuppress the gene expression in the antigen presenting cells throughmiR-142 mediated mRNA degradation, limiting antigen presentation inprofessional APCs (e.g., dendritic cells) and thereby preventingantigen-mediated immune response after gene delivery (see, Annoni A etal., blood, 2009, 114, 5152-5161.

In one example, miRNAs binding sites that are known to be expressed inimmune cells, in particular, the antigen presenting cells, can beengineered into the polynucleotides to suppress the expression of thepolynucleotide in APCs through miRNA mediated RNA degradation, subduingthe antigen-mediated immune response, while the expression of thepolynucleotide is maintained in non-immune cells where the immune cellspecific miRNAs are not expressed.

Many miRNA expression studies have been conducted, and are described inthe art, to profile the differential expression of miRNAs in variouscancer cells/tissues and other diseases. Some miRNAs are abnormallyover-expressed in certain cancer cells and others are under-expressed.For example, miRNAs are differentially expressed in cancer cells(WO2008/154098, US2013/0059015, US2013/0042333, WO2011/157294); cancerstem cells (US2012/0053224); pancreatic cancers and diseases(US2009/0131348, US2011/0171646, US2010/0286232, U.S. Pat. No.8,389,210); asthma and inflammation (U.S. Pat. No. 8,415,096); prostatecancer (US2013/0053264); hepatocellular carcinoma (WO2012/151212,US2012/0329672, WO2008/054828, U.S. Pat. No. 8,252,538); lung cancercells (WO2011/076143, WO2013/033640, WO2009/070653, US2010/0323357);cutaneous T-cell lymphoma (WO2013/011378); colorectal cancer cells(WO2011/0281756, WO2011/076142); cancer positive lymph nodes(WO2009/100430, US2009/0263803); nasopharyngeal carcinoma (EP2112235);chronic obstructive pulmonary disease (US2012/0264626, US2013/0053263);thyroid cancer (WO2013/066678); ovarian cancer cells (US2012/0309645,WO2011/095623); breast cancer cells (WO2008/154098, WO2007/081740,US2012/0214699), leukemia and lymphoma (WO2008/073915, US2009/0092974,US2012/0316081, US2012/0283310, WO2010/018563.

Human Cells

For ameliorating Usher Syndrome Type 2A or any disorder associated withUSH2A, as described and illustrated herein, the principal targets forgene editing are human cells. For example, in the ex vivo methods, thehuman cells can be somatic cells, which after being modified using thetechniques as described, can give rise to differentiated cells, e.g.,photoreceptor cells or retinal progenitor cells. For example, in the invivo methods, the human cells can be photoreceptor cells or retinalprogenitor cells.

By performing gene editing in autologous cells that are derived from andtherefore already completely matched with the patient in need, it ispossible to generate cells that can be safely re-introduced into thepatient, and effectively give rise to a population of cells that can beeffective in ameliorating one or more clinical conditions associatedwith the patient's disease.

Progenitor cells (also referred to as stem cells herein) are capable ofboth proliferation and giving rise to more progenitor cells, these inturn having the ability to generate a large number of mother cells thatcan in turn give rise to differentiated or differentiable daughtercells. The daughter cells themselves can be induced to proliferate andproduce progeny that subsequently differentiate into one or more maturecell types, while also retaining one or more cells with parentaldevelopmental potential. The term “stem cell” refers then, to a cellwith the capacity or potential, under particular circumstances, todifferentiate to a more specialized or differentiated phenotype, andwhich retains the capacity, under certain circumstances, to proliferatewithout substantially differentiating. In one aspect, the termprogenitor or stem cell refers to a generalized mother cell whosedescendants (progeny) specialize, often in different directions, bydifferentiation, e.g., by acquiring completely individual characters, asoccurs in progressive diversification of embryonic cells and tissues.Cellular differentiation is a complex process typically occurringthrough many cell divisions. A differentiated cell can derive from amultipotent cell that itself is derived from a multipotent cell, and soon. While each of these multipotent cells can be considered stem cells,the range of cell types that each can give rise to can varyconsiderably. Some differentiated cells also have the capacity to giverise to cells of greater developmental potential. Such capacity can benatural or can be induced artificially upon treatment with variousfactors. In many biological instances, stem cells can also be“multipotent” because they can produce progeny of more than one distinctcell type, but this is not required for “stem-ness.”

Self-renewal can be another important aspect of the stem cell. Intheory, self-renewal can occur by either of two major mechanisms. Stemcells can divide asymmetrically, with one daughter retaining the stemstate and the other daughter expressing some distinct other specificfunction and phenotype. Alternatively, some of the stem cells in apopulation can divide symmetrically into two stems, thus maintainingsome stem cells in the population as a whole, while other cells in thepopulation give rise to differentiated progeny only. Generally,“progenitor cells” have a cellular phenotype that is more primitive(i.e., is at an earlier step along a developmental pathway orprogression than is a fully differentiated cell). Often, progenitorcells also have significant or very high proliferative potential.Progenitor cells can give rise to multiple distinct differentiated celltypes or to a single differentiated cell type, depending on thedevelopmental pathway and on the environment in which the cells developand differentiate.

In the context of cell ontogeny, the adjective “differentiated,” or“differentiating” is a relative term. A “differentiated cell” is a cellthat has progressed further down the developmental pathway than the cellto which it is being compared. Thus, stem cells can differentiate intolineage-restricted precursor cells (such as a myocyte progenitor cell),which in turn can differentiate into other types of precursor cellsfurther down the pathway (such as a myocyte precursor), and then to anend-stage differentiated cell, such as a myocyte, which plays acharacteristic role in a certain tissue type, and can or cannot retainthe capacity to proliferate further.

Edited Human Cells

Provided herein are methods for editing an IVS40 mutation in a USH2Agene in a human cell. Provided herein are gRNAs for editing an IVS40mutation in a USH2A gene in a human cell.

These methods and/or gRNAs disclosed herein can be used to edit apopulation of human cells. A number of human cells within a cellpopulation sufficient for use in treating a patient can be edited. Forexample, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% of thehuman cells within a cell population can be edited and can be sufficientto use to treat a patient. In other examples, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5%, 1%, or 0.5% of the human cells within a cellpopulation can be edited and can be sufficient to use to treat apatient. In various examples, the edited human cells can be firstselected and cultured to expand the number of edited cells beforeadministering them to a patient.

Induced Pluripotent Stem Cells

The genetically engineered human cells described herein can be inducedpluripotent stem cells (iPSCs). An advantage of using iPSCs is that thecells can be derived from the same subject to which the progenitor cellsare to be administered. That is, a somatic cell can be obtained from asubject, reprogrammed to an induced pluripotent stem cell, and thenre-differentiated into a progenitor cell to be administered to thesubject (e.g., autologous cells). Because the progenitors areessentially derived from an autologous source, the risk of engraftmentrejection or allergic response can be reduced compared to the use ofcells from another subject or group of subjects. In addition, the use ofiPSCs negates the need for cells obtained from an embryonic source.Thus, in one aspect, the stem cells used in the disclosed methods arenot embryonic stem cells.

Although differentiation is generally irreversible under physiologicalcontexts, several methods have been recently developed to reprogramsomatic cells to iPSCs. Exemplary methods are known to those of skill inthe art and are described briefly herein below.

The term “reprogramming” refers to a process that alters or reverses thedifferentiation state of a differentiated cell (e.g., a somatic cell).Stated another way, reprogramming refers to a process of driving thedifferentiation of a cell backwards to a more undifferentiated or moreprimitive type of cell. It should be noted that placing many primarycells in culture can lead to some loss of fully differentiatedcharacteristics. Thus, simply culturing such cells included in the termdifferentiated cells does not render these cells non-differentiatedcells (e.g., undifferentiated cells) or pluripotent cells. Thetransition of a differentiated cell to pluripotency requires areprogramming stimulus beyond the stimuli that lead to partial loss ofdifferentiated character in culture. Reprogrammed cells also have thecharacteristic of the capacity of extended passaging without loss ofgrowth potential, relative to primary cell parents, which generally havecapacity for only a limited number of divisions in culture.

The cell to be reprogrammed can be either partially or terminallydifferentiated prior to reprogramming. Reprogramming can encompasscomplete reversion of the differentiation state of a differentiated cell(e.g., a somatic cell) to a pluripotent state or a multipotent state.Reprogramming can encompass complete or partial reversion of thedifferentiation state of a differentiated cell (e.g., a somatic cell) toan undifferentiated cell (e.g., an embryonic-like cell). Reprogrammingcan result in expression of particular genes by the cells, theexpression of which further contributes to reprogramming. In certainexamples described herein, reprogramming of a differentiated cell (e.g.,a somatic cell) can cause the differentiated cell to assume anundifferentiated state (e.g., is an undifferentiated cell). Theresulting cells are referred to as “reprogrammed cells,” or “inducedpluripotent stem cells (iPSCs or iPS cells).”

Reprogramming can involve alteration, e.g., reversal, of at least someof the heritable patterns of nucleic acid modification (e.g.,methylation), chromatin condensation, epigenetic changes, genomicimprinting, etc., that occur during cellular differentiation.Reprogramming is distinct from simply maintaining the existingundifferentiated state of a cell that is already pluripotent ormaintaining the existing less than fully differentiated state of a cellthat is already a multipotent cell (e.g., a myogenic stem cell).Reprogramming is also distinct from promoting the self-renewal orproliferation of cells that are already pluripotent or multipotent,although the compositions and methods described herein can also be ofuse for such purposes, in some examples.

Many methods are known in the art that can be used to generatepluripotent stem cells from somatic cells. Any such method thatreprograms a somatic cell to the pluripotent phenotype would beappropriate for use in the methods described herein.

Reprogramming methodologies for generating pluripotent cells usingdefined combinations of transcription factors have been described. Mousesomatic cells can be converted to ES cell-like cells with expandeddevelopmental potential by the direct transduction of Oct4, Sox2, Klf4,and c-Myc; see, e.g., Takahashi and Yamanaka, Cell 126(4): 663-76(2006). iPSCs resemble ES cells, as they restore thepluripotency-associated transcriptional circuitry and much of theepigenetic landscape. In addition, mouse iPSCs satisfy all the standardassays for pluripotency: specifically, in vitro differentiation intocell types of the three germ layers, teratoma formation, contribution tochimeras, germline transmission [see, e.g., Maherali and Hochedlinger,Cell Stem Cell. 3(6):595-605 (2008)], and tetraploid complementation.

Human iPSCs can be obtained using similar transduction methods, and thetranscription factor trio, OCT4, SOX2, and NANOG, has been establishedas the core set of transcription factors that govern pluripotency; see,e.g., Budniatzky and Gepstein, Stem Cells Transl Med. 3(4):448-57(2014); Barrett et al., Stem Cells Trans Med 3:1-6 sctm.2014-0121(2014); Focosi et al., Blood Cancer Journal 4: e211 (2014). Theproduction of iPSCs can be achieved by the introduction of nucleic acidsequences encoding stem cell-associated genes into an adult, somaticcell, historically using viral vectors.

iPSCs can be generated or derived from terminally differentiated somaticcells, as well as from adult stem cells, or somatic stem cells. That is,a non-pluripotent progenitor cell can be rendered pluripotent ormultipotent by reprogramming. In such instances, it cannot be necessaryto include as many reprogramming factors as required to reprogram aterminally differentiated cell. Further, reprogramming can be induced bythe non-viral introduction of reprogramming factors, e.g., byintroducing the proteins themselves, or by introducing nucleic acidsthat encode the reprogramming factors, or by introducing messenger RNAsthat upon translation produce the reprogramming factors (see e.g.,Warren et al., Cell Stem Cell, 7(5):618-30 (2010). Reprogramming can beachieved by introducing a combination of nucleic acids encoding stemcell-associated genes, including, for example, Oct-4 (also known asOct-3/4 or Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klf1, Klf2,Klf4, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, and LIN28.Reprogramming using the methods and compositions described herein canfurther comprise introducing one or more of Oct-3/4, a member of the Soxfamily, a member of the Klf family, and a member of the Myc family to asomatic cell. The methods and compositions described herein can furthercomprise introducing one or more of each of Oct-4, Sox2, Nanog, c-MYCand Klf4 for reprogramming. As noted above, the exact method used forreprogramming is not necessarily critical to the methods andcompositions described herein. However, where cells differentiated fromthe reprogrammed cells are to be used in, e.g., human therapy, in oneaspect the reprogramming is not effected by a method that alters thegenome. Thus, in such examples, reprogramming can be achieved, e.g.,without the use of viral or plasmid vectors.

The efficiency of reprogramming (i.e., the number of reprogrammed cells)derived from a population of starting cells can be enhanced by theaddition of various agents, e.g., small molecules, as shown by Shi etal., Cell-Stem Cell 2:525-528 (2008); Huangfu et al., NatureBiotechnology 26(7):795-797 (2008) and Marson et al., Cell-Stem Cell 3:132-135 (2008).

Thus, an agent or combination of agents that enhance the efficiency orrate of induced pluripotent stem cell production can be used in theproduction of patient-specific or disease-specific iPSCs. Somenon-limiting examples of agents that enhance reprogramming efficiencyinclude soluble Wnt, Wnt conditioned media, BIX-01294 (a G9a histonemethyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferaseinhibitors, histone deacetylase (HDAC) inhibitors, valproic acid,5′-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA),vitamin C, and trichostatin (TSA), among others.

Other non-limiting examples of reprogramming enhancing agents include:Suberoylanilide Hydroxamic Acid (SAHA (e.g., MK0683, vorinostat) andother hydroxamic acids), BML-210, Depudecin (e.g., (−)-Depudecin), HCToxin, Nullscript(4-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide),Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VP A)and other short chain fatty acids), Scriptaid, Suramin Sodium,Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate,pivaloyloxymethyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin,Depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994(e.g., N-acetyl dinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA(m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, Tubacin,A-161906, proxamide, oxamflatin, 3-C1-UCHA (e.g.,6-(3-chlorophenylureido)caproic hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP 50. Other reprogrammingenhancing agents include, for example, dominant negative forms of theHDACs (e.g., catalytically inactive forms), siRNA inhibitors of theHDACs, and antibodies that specifically bind to the HDACs. Suchinhibitors are available, e.g., from BIOMOL International, Fukasawa,Merck Biosciences, Novartis, Gloucester Pharmaceuticals, TitanPharmaceuticals, MethylGene, and Sigma Aldrich.

To confirm the induction of pluripotent stem cells for use with themethods described herein, isolated clones can be tested for theexpression of a stem cell marker. Such expression in a cell derived froma somatic cell identifies the cells as induced pluripotent stem cells.Stem cell markers can be selected from the non-limiting group includingSSEA3, SSEA4, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto,Dax1, Zpf296, S1c2a3, Rex1, Utf1, and Nat1. In one case, for example, acell that expresses Oct4 or Nanog is identified as pluripotent. Methodsfor detecting the expression of such markers can include, for example,RT-PCR and immunological methods that detect the presence of the encodedpolypeptides, such as Western blots or flow cytometric analyses.Detection can involve not only RT-PCR, but can also include detection ofprotein markers. Intracellular markers can be best identified viaRT-PCR, or protein detection methods such as immunocytochemistry, whilecell surface markers are readily identified, e.g., byimmunocytochemistry.

The pluripotent stem cell character of isolated cells can be confirmedby tests evaluating the ability of the iPSCs to differentiate into cellsof each of the three germ layers. As one example, teratoma formation innude mice can be used to evaluate the pluripotent character of theisolated clones. The cells can be introduced into nude mice andhistology and/or immunohistochemistry can be performed on a tumorarising from the cells. The growth of a tumor comprising cells from allthree germ layers, for example, further indicates that the cells arepluripotent stem cells.

Retinal Progenitor Cells and Photoreceptor Cells

In some examples, the genetically engineered human cells describedherein are photoreceptor cells or retinal progenitor cells (RPCs). RPCsare multipotent progenitor cells that can give rise to all the sixneurons of the retina as well as the Müller glia. Müller glia are a typeof retinal glial cells and are the major glial component of the retina.Their function is to support the neurons of the retina and to maintainretinal homeostasis and integrity. Müller glia isolated from adult humanretinas have been shown to differentiate into rod photoreceptors.Functional characterization of such Müller glia-derived photoreceptorsby patch-clamp recordings has revealed that their electrical propertiesare comparable to those of adult rods (Giannelli et al., 2011, StemCells, (2):344-56). RPCs are gradually specified into lineage-restrictedprecursor cells during retinogenesis, which then maturate into theterminally differentiated neurons or Müller glia. Fetal-derived humanretinal progenitor cells (hRPCs) exhibit molecular characteristicsindicative of a retinal progenitor state up to the sixth passage. Theydemonstrate a gradual decrease in the percentages of KI67-, SOX2-, andvimentin-positive cells from passages 1 to 6, whereas a sustainedexpression of nestin and PAX6 is seen through passage 6. Microarrayanalysis of passage 1 hRPCs demonstrate the expression of early retinaldevelopmental genes: VIM (vimentin), KI67, NES (nestin), PAX6, SOX2,HESS, GNL3, OTX2, DACH1, SIX6, and CHX10 (VSX2). The hRPCs arefunctional in nature and respond to excitatory neurotransmitters(Schmitt et al., 2009, Investigative Ophthalmology and Visual Sciences.2009; 50(12):5901-8). The outermost region of the retina contains asupportive retinal pigment epithelium (RPE) layer, which maintainsphotoreceptor health by transporting nutrients and recycling shedphotoreceptor parts. The RPE is attached to Bruch's membrane, anextracellular matrix structure at the interface between the choroid andretina. On the other side of the RPE, moving inwards towards theinterior of the eye, there are three layers of neurons: light sensingrod and cone photoreceptors, a middle layer of connecting neurons(amacrine, bipolar and horizontal cells) and the innermost layer ofganglion cells, which transmit signals originating in the photoreceptorlayer through the optic nerve and into the brain. In some aspects, thegenetically engineered human cells described herein are photoreceptorcells, which are specialized types of neurons found in the retina.Photoreceptors convert light into signals that are able to stimulatebiological processes and are responsible for sight. Rods and cones arethe two classic photoreceptor cells that contribute information to thevisual system.

Isolating a Retinal Progenitor Cell and Photoreceptor Cell

Retinal cells, including progenitor cells may be isolated according toany method known in the art. For example, human retinal cells areisolated from fresh surgical specimens. The retinal pigment epithelium(RPE) is separated from the choroid by digesting the tissue with type IVcollagenase and the retinal pigment epithelium patches are cultured.Following the growth of 100-500 cells from the explant, the primarycultures are passaged (Ishida M. et al., Current Eye Research 1998;17(4):392-402) and characterized for expression of RPE markers. Rods areisolated by disruption of the biopsied retina using papain. Precautionsare taken to avoid a harsh disruption and improve cell yield. Theisolated cells are sorted to yield a population of pure rod cells andcharacterized further by immunostaining (Feodorova et al., MethodsX2015; 2:39-46).

In order to isolate cones, neural retina is identified, cut-out, andplaced on 10% gelatin. The inner retinal layers are isolated using alaser. The isolated cone monolayers are cultured for 18 hours andcompared with untreated retinas by light microscopy and transmissionmicroscopy to check for any structural damage. The cells arecharacterized for expression of cone-specific markers (Salchow et al.,Current Eye Research 2001; 22).

In order to isolate retinal progenitor cells, the biopsied retina isminced with dual scalpels and digested enzymatically in an incubator at37° C. The supernatants of the digested cells are centrifuged and thecells are resuspended in cell-free retinal progenitor-conditionedmedium. The cells are transferred to fibronectin-coated tissue cultureflasks containing fresh media and cultured (Klassen et al., Journal ofNeuroscience Research 2004; 77:334-343).

Creating Patient Specific iPSCs

One step of the ex vivo methods of the present disclosure can involvecreating a patient-specific iPS cell, patient-specific iPS cells, or apatient-specific iPS cell line. There are many established methods inthe art for creating patient specific iPS cells, as described inTakahashi and Yamanaka 2006; Takahashi, Tanabe et al. 2007. For example,the creating step can comprise: a) isolating a somatic cell, such as askin cell or fibroblast, from the patient; and b) introducing a set ofpluripotency-associated genes into the somatic cell in order to inducethe cell to become a pluripotent stem cell. The set ofpluripotency-associated genes can be one or more of the genes selectedfrom the group consisting of OCT4, SOX1, SOX2, SOX3, SOX15, SOX18,NANOG, KLF1, KLF2, KLF4, KLF5, c-MYC, n-MYC, REM2, TERT and LIN28.

Performing a Biopsy or Aspirate of the Patient's Bone Marrow

A biopsy or aspirate is a sample of tissue or fluid taken from the body.There are many different kinds of biopsies or aspirates. Nearly all ofthem involve using a sharp tool to remove a small amount of tissue. Ifthe biopsy will be on the skin or other sensitive area, numbing medicinecan be applied first. A biopsy or aspirate can be performed according toany of the known methods in the art. For example, in a bone marrowaspirate, a large needle is used to enter the pelvis bone to collectbone marrow.

Isolating a Mesenchymal Stem Cell

Mesenchymal stem cells can be isolated according to any method known inthe art, such as from a patient's bone marrow or peripheral blood. Forexample, marrow aspirate can be collected into a syringe with heparin.Cells can be washed and centrifuged on a Percoll™ density gradient.Cells, such as blood cells, liver cells, interstitial cells,macrophages, mast cells, and thymocytes, can be separated using densitygradient centrifugation media, Percoll™. The cells can then be culturedin Dulbecco's modified Eagle's medium (DMEM) (low glucose) containing10% fetal bovine serum (FBS) (Pittinger M F, Mackay A M, Beck S C etal., Science 1999; 284:143-147).

Differentiation of Genome-Edited iPSCs into Other Cell Types

Another step of the ex vivo methods of the present disclosure cancomprise differentiating the genome-edited iPSCs into photoreceptorcells or retinal progenitor cells. The differentiating step may beperformed according to any method known in the art. For example, iPSCscan be used to generate retinal organioids and photoreceptors asdescribed in the art (Phillips et al., Stem Cells, June 2014, 32(6):pgs. 1480-1492; Zhong et al. Nat. Commun., 2014, 5: pg 4047; Tucker etal., PLoS One, April 2011, 6(4): e18992). For example, hiPSC aredifferentiated into retinal progenitor cells using various treatments,including Wnt, Nodal, and Notch pathway inhibitors (Noggin, Dk1, LeftyA,and DAPT) and other growth factors. The retinal progenitor cells arefurther differentiated into photoreceptor cells, the treatmentincluding: exposure to native retinal cells in coculture systems, RX+ orMitf+ by subsequent treatment with retinoic acid and taurine, orexposure to several exogenous factors including Noggin, Dkk1, DAPT, andinsulin-like growth factor (Yang et al., Stem Cells International 2016).

Differentiation of Genome-Edited Mesenchymal Stem Cells intoPhotoreceptor Cells or Retinal Progenitor Cells

Another step of the ex vivo methods of the present disclosure cancomprise differentiating the genome-edited mesenchymal stem cells intophotoreceptor cells or retinal progenitor cells. The differentiatingstep can be performed according to any method known in the art.

Implanting Cells into Patients

Another step of the ex vivo methods of the present disclosure cancomprise implanting the photoreceptor cells or retinal progenitor cellsinto patients. This implanting step can be accomplished using any methodof implantation known in the art. For example, the genetically modifiedcells can be injected directly in the patient's blood or otherwiseadministered to the patient.

Another step of the ex vivo methods of the invention involves implantingthe photoreceptor cells or retinal progenitor cells into patients. Thisimplanting step can be accomplished using any method of implantationknown in the art. For example, the genetically modified cells can beinjected directly in the patient's eye or otherwise administered to thepatient.

Genetically Modified Cells

The term “genetically modified cell” refers to a cell that comprises atleast one genetic modification introduced by genome editing (e.g., usingthe CRISPR/Cas9/Cpf1 system). In some ex vivo examples herein, thegenetically modified cell can be genetically modified progenitor cell.In some in vivo examples herein, the genetically modified cell can be agenetically modified photoreceptor cell or retinal progenitor cell. Agenetically modified cell comprising an exogenous genome-targetingnucleic acid and/or an exogenous nucleic acid encoding agenome-targeting nucleic acid is contemplated herein.

The term “control treated population” describes a population of cellsthat has been treated with identical media, viral induction, nucleicacid sequences, temperature, confluency, flask size, pH, etc., with theexception of the addition of the genome editing components. Any methodknown in the art can be used to measure restoration of USH2A gene orusherin protein expression or activity, for example Western Blotanalysis of the usherin protein or real time PCR for quantifying USH2AmRNA.

The term “isolated cell” refers to a cell that has been removed from anorganism in which it was originally found, or a descendant of such acell. Optionally, the cell can be cultured in vitro, e.g., under definedconditions or in the presence of other cells. Optionally, the cell canbe later introduced into a second organism or re-introduced into theorganism from which it (or the cell from which it is descended) wasisolated.

The term “isolated population” with respect to an isolated population ofcells refers to a population of cells that has been removed andseparated from a mixed or heterogeneous population of cells. In somecases, the isolated population can be a substantially pure population ofcells, as compared to the heterogeneous population from which the cellswere isolated or enriched. In some cases, the isolated population can bean isolated population of human progenitor cells, e.g., a substantiallypure population of human progenitor cells, as compared to aheterogeneous population of cells comprising human progenitor cells andcells from which the human progenitor cells were derived.

The term “substantially enhanced,” with respect to a particular cellpopulation, refers to a population of cells in which the occurrence of aparticular type of cell is increased relative to pre-existing orreference levels, by at least 2-fold, at least 3-, at least 4-, at least5-, at least 6-, at least 7-, at least 8-, at least 9, at least 10-, atleast 20-, at least 50-, at least 100-, at least 400-, at least 1000-,at least 5000-, at least 20000-, at least 100000- or more folddepending, e.g., on the desired levels of such cells for amelioratingUsher Syndrome Type 2A.

The term “substantially enriched” with respect to a particular cellpopulation, refers to a population of cells that is at least about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70% or morewith respect to the cells making up a total cell population.

The terms “substantially pure” with respect to a particular cellpopulation, refers to a population of cells that is at least about 75%,at least about 85%, at least about 90%, or at least about 95% pure, withrespect to the cells making up a total cell population. That is, theterms “substantially pure” or “essentially purified,” with regard to apopulation of progenitor cells, refers to a population of cells thatcontain fewer than about 20%, about 15%, about 10%, about 9%, about 8%,about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, orless than 1%, of cells that are not progenitor cells as defined by theterms herein.

Delivery

Guide RNA polynucleotides (RNA or DNA) and/or endonucleasepolynucleotide(s) (RNA or DNA) can be delivered by viral or non-viraldelivery vehicles known in the art. Alternatively, endonucleasepolypeptide(s) can be delivered by viral or non-viral delivery vehiclesknown in the art, such as electroporation or lipid nanoparticles. Infurther alternative aspects, the DNA endonuclease can be delivered asone or more polypeptides, either alone or pre-complexed with one or moreguide RNAs, or one or more crRNA together with a tracrRNA.

Polynucleotides can be delivered by non-viral delivery vehiclesincluding, but not limited to, nanoparticles, liposomes,ribonucleoproteins, positively charged peptides, small moleculeRNA-conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes.Some exemplary non-viral delivery vehicles are described in Peer andLieberman, Gene Therapy, 18: 1127-1133 (2011) (which focuses onnon-viral delivery vehicles for siRNA that are also useful for deliveryof other polynucleotides).

Polynucleotides, such as guide RNA, sgRNA, and mRNA encoding anendonuclease, can be delivered to a cell or a patient by a lipidnanoparticle (LNP).

A LNP refers to any particle having a diameter of less than 1000 nm, 500nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.Alternatively, a nanoparticle can range in size from 1-1000 nm, 1-500nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

LNPs can be made from cationic, anionic, or neutral lipids. Neutrallipids, such as the fusogenic phospholipid DOPE or the membranecomponent cholesterol, can be included in LNPs as ‘helper lipids’ toenhance transfection activity and nanoparticle stability. Limitations ofcationic lipids include low efficacy owing to poor stability and rapidclearance, as well as the generation of inflammatory oranti-inflammatory responses.

LNPs can also be comprised of hydrophobic lipids, hydrophilic lipids, orboth hydrophobic and hydrophilic lipids.

Any lipid or combination of lipids that are known in the art can be usedto produce a LNP. Examples of lipids used to produce LNPs are: DOTMA,DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol,GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG).Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2-DMA (KC2),DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids are:

DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are:PEG-DMG, PEG-CerC14, and PEG-CerC20.

The lipids can be combined in any number of molar ratios to produce aLNP. In addition, the polynucleotide(s) can be combined with lipid(s) ina wide range of molar ratios to produce a LNP.

As stated previously, the site-directed polypeptide and genome-targetingnucleic acid can each be administered separately to a cell or a patient.On the other hand, the site-directed polypeptide can be pre-complexedwith one or more guide RNAs, or one or more crRNA together with atracrRNA. The pre-complexed material can then be administered to a cellor a patient. Such pre-complexed material is known as aribonucleoprotein particle (RNP).

RNA is capable of forming specific interactions with RNA or DNA. Whilethis property is exploited in many biological processes, it also comeswith the risk of promiscuous interactions in a nucleic acid-richcellular environment. One solution to this problem is the formation ofribonucleoprotein particles (RNPs), in which the RNA is pre-complexedwith an endonuclease. Another benefit of the RNP is protection of theRNA from degradation.

The endonuclease in the RNP can be modified or unmodified. Likewise, thegRNA, crRNA, tracrRNA, or sgRNA can be modified or unmodified. Numerousmodifications are known in the art and can be used.

The endonuclease and sgRNA can be generally combined in a 1:1 molarratio.

Alternatively, the endonuclease, crRNA and tracrRNA can be generallycombined in a 1:1:1 molar ratio. However, a wide range of molar ratioscan be used to produce a RNP.

AAV (Adeno Associated Virus)

A recombinant adeno-associated virus (AAV) vector can be used fordelivery. Techniques to produce rAAV particles, in which an AAV genometo be packaged that includes the polynucleotide to be delivered, rep andcap genes, and helper virus functions are provided to a cell arestandard in the art. Production of rAAV typically requires that thefollowing components are present within a single cell (denoted herein asa packaging cell): a rAAV genome, AAV rep and cap genes separate from(i.e., not in) the rAAV genome, and helper virus functions. The AAV repand cap genes can be from any AAV serotype for which recombinant viruscan be derived, and can be from a different AAV serotype than the rAAVgenome ITRs, including, but not limited to, AAV serotypes describedherein. Production of pseudotyped rAAV is disclosed in, for example,international patent application publication number WO 01/83692.

AAV Serotypes

AAV particles packaging polynucleotides encoding compositions of thepresent disclosure, e.g., endonucleases, donor sequences, or RNA guidemolecules, of the present disclosure can comprise or be derived from anynatural or recombinant AAV serotype. According to the presentdisclosure, the AAV particles can utilize or be based on a serotypeselected from any of the following serotypes, and variants thereofincluding but not limited to AAV1, AAV10, AAV106.1/hu.37, AAV11,AAV114.3/hu.40, AAV12, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.1/hu.43,AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54,AAV145.6/hu.55, AAV16.12/hu.11, AAV16.3, AAV16.8/hu.10, AAV161.10/hu.60,AAV161.6/hu.61, AAV1-7/rh.48, AAV1-8/rh.49, AAV2, AAV2.5T,AAV2-15/rh.62, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6,AAV223.7, AAV2-3/rh.61, AAV24.1, AAV2-4/rh.50, AAV2-5/rh.51, AAV27.3,AAV29.3/bb.1, AAV29.5/bb.2, AAV2G9, AAV-2-pre-miRNA-101, AAV3,AAV3.1/hu.6, AAV3.1/hu.9, AAV3-11/rh.53, AAV3-3, AAV33.12/hu.17,AAV33.4/hu.15, AAV33.8/hu.16, AAV3-9/rh.52, AAV3a, AAV3b, AAV4,AAV4-19/rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13,AAV42-15, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a,AAV42-5b, AAV42-6b, AAV42-8, AAV42-aa, AAV43-1, AAV43-12, AAV43-20,AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1, AAV44.2,AAV44.5, AAV46.2/hu.28, AAV46.6/hu.29, AAV4-8/r11.64, AAV4-8/rh.64,AAV4-9/rh.54, AAV5, AAV52.1/hu.20, AAV52/hu.19, AAV5-22/rh.58,AAV5-3/rh.57, AAV54.1/hu.21, AAV54.2/hu.22, AAV54.4R/hu.27,AAV54.5/hu.23, AAV54.7/hu.24, AAV58.2/hu.25, AAV6, AAV6.1, AAV6.1.2,AAV6.2, AAV7, AAV7.2, AAV7.3/hu.7, AAV8, AAV-8b, AAV-8h, AAV9, AAV9.11,AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84,AAV9.9, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAV-b, AAVC1, AAVC2, AAVC5,AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1,AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAV-DJ, AAV-DJ8, AAVF3, AAVF5,AAV-h, AAVH-1/hu.1, AAVH2, AAVH-5/hu.3, AAVH6, AAVhE1.1, AAVhER1.14,AAVhEr1.16, AAVhEr1.18, AAVhER1.23, AAVhEr1.35, AAVhEr1.36, AAVhEr1.5,AAVhEr1.7, AAVhEr1.8, AAVhEr2.16, AAVhEr2.29, AAVhEr2.30, AAVhEr2.31,AAVhEr2.36, AAVhEr2.4, AAVhEr3.1, AAVhu.1, AAVhu.10, AAVhu.11, AAVhu.11,AAVhu.12, AAVhu.13, AAVhu.14/9, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18,AAVhu.19, AAVhu.2, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24,AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.3, AAVhu.31,AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.4, AAVhu.40,AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2,AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1,AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.5, AAVhu.51, AAVhu.52, AAVhu.53,AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.6, AAVhu.60,AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.7, AAVhu.8,AAVhu.9, AAVhu.t 19, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39,AAVLG-9/hu.39, AAV-LK01, AAV-LK02, AAVLK03, AAV-LK03, AAV-LK04,AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11,AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK17, AAV-LK18, AAV-LK19,AAVN721-8/rh.43, AAV-PAEC, AAV-PAEC11, AAV-PAEC12, AAV-PAEC2, AAV-PAEC4,AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAVpi.1, AAVpi.2, AAVpi.3, AAVrh.10,AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19,AAVrh.2, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25,AAVrh.2R, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36,AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.44,AAVrh.45, AAVrh.46, AAVrh.47, AAVrh.48, AAVrh.48, AAVrh.48.1,AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.50, AAVrh.51, AAVrh.52,AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59,AAVrh.60, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64R1, AAVrh.64R2,AAVrh.65, AAVrh.67, AAVrh.68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73,AAVrh.74, AAVrh.8, AAVrh.8R, AAVrh8R, AAVrh8R A586R mutant, AAVrh8RR533A mutant, BAAV, BNP61 AAV, BNP62 AAV, BNP63 AAV, bovine AAV, caprineAAV, Japanese AAV 10, true type AAV (ttAAV), UPENN AAV 10, AAV-LK16,AAAV, AAV Shuffle 100-1, AAV Shuffle 100-2, AAV Shuffle 100-3, AAVShuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAVSM 100-10, AAV SM 100-3, AAV SM 10-1, AAV SM 10-2, and/or AAV SM 10-8.

In some examples, the AAV serotype can be, or have, a mutation in theAAV9 sequence as described by N Pulicherla et al. (Molecular Therapy19(6):1070-1078 (2011), such as but not limited to, AAV9.9, AAV9.11,AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84.

In some examples, the AAV serotype can be, or have, a sequence asdescribed in U.S. Pat. No. 6,156,303, such as, but not limited to, AAV3B(SEQ ID NO: 1 and 10 of U.S. Pat. No. 6,156,303), AAV6 (SEQ ID NO: 2, 7and 11 of U.S. Pat. No. 6,156,303), AAV2 (SEQ ID NO: 3 and 8 of U.S.Pat. No. 6,156,303), AAV3A (SEQ ID NO: 4 and 9, of U.S. Pat. No.6,156,303), or derivatives thereof.

In some examples, the serotype can be AAVDJ or a variant thereof, suchas AAVDJ8 (or AAV-DJ8), as described by Grimm et al. (Journal ofVirology 82(12): 5887-5911 (2008)). The amino acid sequence of AAVDJ8can comprise two or more mutations in order to remove the heparinbinding domain (HBD). As a non-limiting example, the AAV-DJ sequencedescribed as SEQ ID NO: 1 in U.S. Pat. No. 7,588,772, can comprise twomutations: (1) R587Q where arginine (R; Arg) at amino acid 587 ischanged to glutamine (Q; Gln) and (2) R590T where arginine (R; Arg) atamino acid 590 is changed to threonine (T; Thr). As another non-limitingexample, can comprise three mutations: (1) K406R where lysine (K; Lys)at amino acid 406 is changed to arginine (R; Arg), (2) R587Q wherearginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln) and(3) R590T where arginine (R; Arg) at amino acid 590 is changed tothreonine (T; Thr).

In some examples, the AAV serotype can be, or have, a sequence asdescribed in International Publication No. WO2015121501, such as, butnot limited to, true type AAV (ttAAV) (SEQ ID NO: 2 of WO2015121501),“UPenn AAV10” (SEQ ID NO: 8 of WO2015121501), “Japanese AAV10” (SEQ IDNO: 9 of WO2015121501), or variants thereof.

According to the present disclosure, AAV capsid serotype selection oruse can be from a variety of species. In one example, the AAV can be anavian AAV (AAAV). The AAAV serotype can be, or have, a sequence asdescribed in U.S. Pat. No. 9,238,800, such as, but not limited to, AAAV(SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, and 14 of U.S. Pat. No. 9,238,800),or variants thereof.

In one example, the AAV can be a bovine AAV (BAAV). The BAAV serotypecan be, or have, a sequence as described in U.S. Pat. No. 9,193,769,such as, but not limited to, BAAV (SEQ ID NO: 1 and 6 of U.S. Pat. No.9,193,769), or variants thereof. The BAAV serotype can be or have asequence as described in U.S. Pat. No. 7,427,396, such as, but notlimited to, BAAV (SEQ ID NO: 5 and 6 of U.S. Pat. No. 7,427,396), orvariants thereof.

In one example, the AAV can be a caprine AAV. The caprine AAV serotypecan be, or have, a sequence as described in U.S. Pat. No. 7,427,396,such as, but not limited to, caprine AAV (SEQ ID NO: 3 of U.S. Pat. No.7,427,396), or variants thereof.

In other examples the AAV can be engineered as a hybrid AAV from two ormore parental serotypes. In one example, the AAV can be AAV2G9 whichcomprises sequences from AAV2 and AAV9. The AAV2G9 AAV serotype can be,or have, a sequence as described in United States Patent Publication No.US20160017005.

In one example, the AAV can be a serotype generated by the AAV9 capsidlibrary with mutations in amino acids 390-627 (VP1 numbering) asdescribed by Pulicherla et al. (Molecular Therapy 19(6):1070-1078(2011). The serotype and corresponding nucleotide and amino acidsubstitutions can be, but is not limited to, AAV9.1 (G1594C; D532H),AAV6.2 (T1418A and T1436X; V473D and I479K), AAV9.3 (T1238A; F413Y),AAV9.4 (T1250C and A1617T; F417S), AAV9.5 (A1235G, A1314T, A1642G,C1760T; Q412R, T548A, A587V), AAV9.6 (T1231A; F411I), AAV9.9 (G1203A,G1785T; W595C), AAV9.10 (A1500G, T1676C; M559T), AAV9.11 (A1425T,A1702C, A1769T; T568P, Q590L), AAV9.13 (A1369C, A1720T; N457H, T574S),AAV9.14 (T1340A, T1362C, T1560C, G1713A; L447H), AAV9.16 (A1775T;Q592L), AAV9.24 (T1507C, T1521G; W503R), AAV9.26 (A1337G, A1769C; Y446C,Q590P), AAV9.33 (A1667C; D556A), AAV9.34 (A1534G, C1794T; N512D),AAV9.35 (A1289T, T1450A, C1494T, A1515T, C1794A, G1816A; Q430L, Y484N,N98K, V6061), AAV9.40 (A1694T, E565V), AAV9.41 (A1348T, T1362C; T450S),AAV9.44 (A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T;N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W509R, L517V),9.47 (G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T582I),AAV9.48 (C1445T, A1736T; P482L, Q579L), AAV9.50 (A1638T, C1683T, T1805A;Q546H, L602H), AAV9.53 (G1301A, A1405C, C1664T, G1811T; R134Q, S469R,A555V, G604V), AAV9.54 (C1531A, T1609A; L511I, L537M), AAV9.55 (T1605A;F535L), AAV9.58 (C1475T, C1579A; T492I, H527N), AAV.59 (T1336C; Y446H),AAV9.61 (A1493T; N498I), AAV9.64 (C1531A, A1617T; L511I), AAV9.65(C1335T, T1530C, C1568A; A523D), AAV9.68 (C1510A; P504T), AAV9.80(G1441A; G481R), AAV9.83 (C1402A, A1500T; P468T, E500D), AAV9.87(T1464C, T1468C; S490P), AAV9.90 (A1196T; Y399F), AAV9.91 (T1316G,A1583T, C1782G, T1806C; L439R, K528I), AAV9.93 (A1273G, A1421G, A1638C,C1712T, G1732A, A1744T, A1832T; S425G, Q474R, Q546H, P571L, G578R,T582S, D611V), AAV9.94 (A1675T; M559L) and AAV9.95 (T1605A; F535L).

In one example, the AAV can be a serotype comprising at least one AAVcapsid CD8+ T-cell epitope. As a non-limiting example, the serotype canbe AAV1, AAV2 or AAV8.

In one example, the AAV can be a variant, such as PHP.A or PHP.B asdescribed in Deverman. 2016. Nature Biotechnology. 34(2): 204-209.

In one example, the AAV can be a serotype selected from any of thosefound in SEQ ID NOs: 4697-5265 and Table 3.

In one example, the AAV can be encoded by a sequence, fragment orvariant as described in SEQ ID NOs: 4697-5265 and Table 3.

A method of generating a packaging cell involves creating a cell linethat stably expresses all of the necessary components for AAV particleproduction. For example, a plasmid (or multiple plasmids) comprising arAAV genome lacking AAV rep and cap genes, AAV rep and cap genesseparate from the rAAV genome, and a selectable marker, such as aneomycin resistance gene, are integrated into the genome of a cell. AAVgenomes have been introduced into bacterial plasmids by procedures suchas GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA,79:2077-2081), addition of synthetic linkers containing restrictionendonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) orby direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem.,259:4661-4666). The packaging cell line can then be infected with ahelper virus, such as adenovirus. The advantages of this method are thatthe cells are selectable and are suitable for large-scale production ofrAAV. Other examples of suitable methods employ adenovirus orbaculovirus, rather than plasmids, to introduce rAAV genomes and/or repand cap genes into packaging cells.

General principles of rAAV production are reviewed in, for example,Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka,1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Variousapproaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072(1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984);Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J.Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol.,7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat.No. 5,173,414; WO 95/13365 and corresponding U.S. Pat. No. 5,658,776; WO95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243(PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark etal. (1996) Gene Therapy 3:1124-1132; U.S. Pat. Nos. 5,786,211;5,871,982; and 6,258,595.

AAV vector serotypes can be matched to target cell types. For example,the following exemplary cell types can be transduced by the indicatedAAV serotypes among others.

TABLE 3 Tissue/Cell Types and Serotypes Tissue/Cell Type Serotype LiverAAV3, AA5, AAV8, AAV9 Skeletal muscle AAV1, AAV7, AAV6, AAV8, AAV9Central nervous system AAV1, AAV4, AAV5, AAV8, AAV9 RPE AAV5, AAV4,AAV2, AAV8, AAV9 AAVrh8r Photoreceptor cells AAV5, AAV8, AAV9, AAVrh8RLung AAV9, AAV5 Heart AAV8 Pancreas AAV8 Kidney AAV2, AAV8

In addition to adeno-associated viral vectors, other viral vectors canbe used. Such viral vectors include, but are not limited to, lentivirus,alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, EpsteinBarr virus, papovavirusr, poxvirus, vaccinia virus, and herpes simplexvirus.

In some cases, Cas9 mRNA, sgRNA targeting one or two loci in USH2A gene,and donor DNA can each be separately formulated into lipidnanoparticles, or are all co-formulated into one lipid nanoparticle.

In some cases, Cas9 mRNA can be formulated in a lipid nanoparticle,while sgRNA and donor DNA can be delivered in an AAV vector.

Options are available to deliver the Cas9 nuclease as a DNA plasmid, asmRNA or as a protein. The guide RNA can be expressed from the same DNA,or can also be delivered as an RNA. The RNA can be chemically modifiedto alter or improve its half-life, or decrease the likelihood or degreeof immune response. The endonuclease protein can be complexed with thegRNA prior to delivery. Viral vectors allow efficient delivery; splitversions of Cas9 and smaller orthologs of Cas9 can be packaged in AAV,as can donors for HDR. A range of non-viral delivery methods also existthat can deliver each of these components, or non-viral and viralmethods can be employed in tandem. For example, nanoparticles can beused to deliver the protein and guide RNA, while AAV can be used todeliver a donor DNA.

Lentivirus

In some aspects, lentiviral vectors or particles can be used as deliveryvehicles. Lentiviruses are subgroup of the Retroviridae family ofviruses. Lentiviral particles are able to integrate their geneticmaterial into the genome of a target/host cell. Examples of lentivirusinclude the Human Immunodeficiency Viruses: HIV-1 and HIV-2, JembranaDisease Virus (JDV), equine infectious anemia virus (EIAV), equineinfectious anemia virus, visna-maedi and caprine arthritis encephalitisvirus (CAEV), the Simian Immunodeficiency Virus (SIV), felineimmunodeficiency virus (FIV), bovine immunodeficiency virus (BIV). LV'sare capable of infecting both dividing and non-dividing cells due totheir unique ability to pass through a target cell's intact nuclearmembrane Greenberg et al., University of Berkeley, Calif.; 2006).Lentiviral particles that form the gene delivery vehicle are replicationdefective and are generated by attenuating the HIV virulence genes. Forexample, the genes Vpu, Vpr, Nef, Env, and Tat are excised making thevector biologically safe. Lentiviral vehicles, for example, derived fromHIV-1/HIV-2 can mediate the efficient delivery, integration andlong-term expression of transgenes into non-dividing cells. As usedherein, the term “recombinant” refers to a vector or other nucleic acidcontaining both lentiviral sequences and non-lentiviral retroviralsequences.

In order to produce a lentivirus that is capable of infecting hostcells, three types of vectors need to be co-expressed in virus producingcells: a backbone vector containing the transgene of interests andself-inactivating 3′-LTR regions, one construct expressing viralstructure proteins, and one vector encoding vesicular stomatitis virusglycoprotein (VSVG) for encapsulation (Naldini, L. et al., Science 1996;272, 263-267). Separation of the Rev gene from other structural genesfurther increases the biosafety by reducing the possibility of reverserecombination. Cell lines that can be used to produce high-titerlentiviral particles may include, but are not limited to 293T cells,293FT cells, and 293SF-3F6 cells (Witting et al., Human Gene Therapy,2012; 23: 243-249; Ansorge et al., Journal of Genetic Medicine, 2009;11: 868-876).

Methods for generating recombinant lentiviral particles are discussed inthe art, for example, WO 2013076309 (PCT/EP2012/073645); WO 2009153563(PCT/GB2009/001527); U.S. Pat. Nos. 7,629,153; and 6,808,905.

Cell types such as photoreceptors, retinal pigment epithelium, andganglion cells have been successfully targeted with lentivirus (LV)vector. The efficiency of delivery to photoreceptors and ganglion cellsis significantly higher with AAV than LV vectors.

Pharmaceutically Acceptable Carriers

The ex vivo methods of administering progenitor cells to a subjectcontemplated herein involve the use of therapeutic compositionscomprising progenitor cells.

Therapeutic compositions can contain a physiologically tolerable carriertogether with the cell composition, and optionally at least oneadditional bioactive agent as described herein, dissolved or dispersedtherein as an active ingredient. In some cases, the therapeuticcomposition is not substantially immunogenic when administered to amammal or human patient for therapeutic purposes, unless so desired.

In general, the progenitor cells described herein can be administered asa suspension with a pharmaceutically acceptable carrier. One of skill inthe art will recognize that a pharmaceutically acceptable carrier to beused in a cell composition will not include buffers, compounds,cryopreservation agents, preservatives, or other agents in amounts thatsubstantially interfere with the viability of the cells to be deliveredto the subject. A formulation comprising cells can include e.g., osmoticbuffers that permit cell membrane integrity to be maintained, andoptionally, nutrients to maintain cell viability or enhance engraftmentupon administration. Such formulations and suspensions are known tothose of skill in the art and/or can be adapted for use with theprogenitor cells, as described herein, using routine experimentation.

A cell composition can also be emulsified or presented as a liposomecomposition, provided that the emulsification procedure does notadversely affect cell viability. The cells and any other activeingredient can be mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient, and in amountssuitable for use in the therapeutic methods described herein.

Additional agents included in a cell composition can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids, such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases, such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH value, physiological salineor both, such as phosphate-buffered saline. Still further, aqueouscarriers can contain more than one buffer salt, as well as salts such assodium and potassium chlorides, dextrose, polyethylene glycol and othersolutes. Liquid compositions can also contain liquid phases in additionto and to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, andwater-oil emulsions. The amount of an active compound used in the cellcompositions that is effective in the treatment of a particular disorderor condition can depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques.

Guide RNA Formulation

Guide RNAs of the present disclosure can be formulated withpharmaceutically acceptable excipients such as carriers, solvents,stabilizers, adjuvants, diluents, etc., depending upon the particularmode of administration and dosage form. Guide RNA compositions can beformulated to achieve a physiologically compatible pH, and range from apH of about 3 to a pH of about 11, about pH 3 to about pH 7, dependingon the formulation and route of administration. In some cases, the pHcan be adjusted to a range from about pH 5.0 to about pH 8. In somecases, the compositions can comprise a therapeutically effective amountof at least one compound as described herein, together with one or morepharmaceutically acceptable excipients. Optionally, the compositions cancomprise a combination of the compounds described herein, or can includea second active ingredient useful in the treatment or prevention ofbacterial growth (for example and without limitation, anti-bacterial oranti-microbial agents), or can include a combination of reagents of thepresent disclosure.

Suitable excipients include, for example, carrier molecules that includelarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, and inactive virus particles. Otherexemplary excipients can include antioxidants (for example and withoutlimitation, ascorbic acid), chelating agents (for example and withoutlimitation, EDTA), carbohydrates (for example and without limitation,dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose),stearic acid, liquids (for example and without limitation, oils, water,saline, glycerol and ethanol), wetting or emulsifying agents, pHbuffering substances, and the like.

Administration & Efficacy

The terms “administering,” “introducing” and “transplanting” can be usedinterchangeably in the context of the placement of cells, e.g.,progenitor cells, into a subject, by a method or route that results inat least partial localization of the introduced cells at a desired site,such as a site of injury or repair, such that a desired effect(s) isproduced. The cells, e.g., progenitor cells, or their differentiatedprogeny can be administered by any appropriate route that results indelivery to a desired location in the subject where at least a portionof the implanted cells or components of the cells remain viable. Theperiod of viability of the cells after administration to a subject canbe as short as a few hours, e.g., twenty-four hours, to a few days, toas long as several years, or even the life time of the patient, i.e.,long-term engraftment. For example, in some aspects described herein, aneffective amount of photoreceptor cells or retinal progenitor cells isadministered via a systemic route of administration, such as anintraperitoneal or intravenous route.

The terms “administering,” “introducing” and “transplanting” can also beused interchangeably in the context of the placement of at least one ofa gRNA, sgRNA, and an endonuclease into a subject, by a method or routethat results in at least partial localization of the introduced gRNA,sgRNA, and/or endonuclease at a desired site, such as a site of injuryor repair, such that a desired effect(s) is produced. The gRNA, sgRNA,and/or endonuclease can be administered by any appropriate route thatresults in delivery to a desired location in the subject.

The terms “individual,” “subject,” “host” and “patient” are usedinterchangeably herein and refer to any subject for whom diagnosis,treatment or therapy is desired. In some aspects, the subject is amammal. In some aspects, the subject is a human being.

When provided prophylactically, progenitor cells described herein can beadministered to a subject in advance of any symptom of Usher SyndromeType 2A. Accordingly, the prophylactic administration of a progenitorcell population serves to prevent Usher Syndrome Type 2A.

A progenitor cell population being administered according to the methodsdescribed herein can comprise allogeneic progenitor cells obtained fromone or more donors. Such progenitors can be of any cellular or tissueorigin, e.g., liver, muscle, cardiac, etc. “Allogeneic” refers to aprogenitor cell or biological samples comprising progenitor cellsobtained from one or more different donors of the same species, wherethe genes at one or more loci are not identical. For example, aphotoreceptor or retinal progenitor cell population being administeredto a subject can be derived from one more unrelated donor subjects, orfrom one or more non-identical siblings. In some cases, syngeneicprogenitor cell populations can be used, such as those obtained fromgenetically identical animals, or from identical twins. The progenitorcells can be autologous cells; that is, the progenitor cells areobtained or isolated from a subject and administered to the samesubject, i.e., the donor and recipient are the same.

The term “effective amount” refers to the amount of a population ofprogenitor cells or their progeny needed to prevent or alleviate atleast one or more signs or symptoms of Usher Syndrome Type 2A, andrelates to a sufficient amount of a composition to provide the desiredeffect, e.g., to treat a subject having Usher Syndrome Type 2A. The term“therapeutically effective amount” therefore refers to an amount ofprogenitor cells or a composition comprising progenitor cells that issufficient to promote a particular effect when administered to a typicalsubject, such as one who has or is at risk for Usher Syndrome Type 2A.An effective amount would also include an amount sufficient to preventor delay the development of a symptom of the disease, alter the courseof a symptom of the disease (for example but not limited to, slow theprogression of a symptom of the disease), or reverse a symptom of thedisease. It is understood that for any given case, an appropriate“effective amount” can be determined by one of ordinary skill in the artusing routine experimentation.

For use in the various aspects described herein, an effective amount ofprogenitor cells comprises at least 10² progenitor cells, at least 5×10²progenitor cells, at least 10³ progenitor cells, at least 5×10³progenitor cells, at least 10⁴ progenitor cells, at least 5×10⁴progenitor cells, at least 10⁵ progenitor cells, at least 2×10⁵progenitor cells, at least 3×10⁵ progenitor cells, at least 4×10⁵progenitor cells, at least 5×10⁵ progenitor cells, at least 6×10⁵progenitor cells, at least 7×10⁵ progenitor cells, at least 8×10⁵progenitor cells, at least 9×10⁵ progenitor cells, at least 1×10⁶progenitor cells, at least 2×10⁶ progenitor cells, at least 3×10⁶progenitor cells, at least 4×10⁶ progenitor cells, at least 5×10⁶progenitor cells, at least 6×10⁶ progenitor cells, at least 7×10⁶progenitor cells, at least 8×10⁶ progenitor cells, at least 9×10⁶progenitor cells, or multiples thereof. The progenitor cells can bederived from one or more donors, or can be obtained from an autologoussource. In some examples described herein, the progenitor cells can beexpanded in culture prior to administration to a subject in needthereof.

Modest and incremental increases in the levels of functional usherinprotein expressed in cells of patients having Usher Syndrome Type 2A canbe beneficial for ameliorating one or more symptoms of the disease, forincreasing long-term survival, and/or for reducing side effectsassociated with other treatments. Upon administration of such cells tohuman patients, the presence of progenitors that are producing increasedlevels of functional usherin protein is beneficial. In some cases,effective treatment of a subject gives rise to at least about 3%, 5% or7% functional usherin protein relative to total usherin in the treatedsubject. In some examples, functional usherin will be at least about 10%of total usherin. In some examples, functional usherin protein will beat least about 20% to 30% of total usherin protein. Similarly, theintroduction of even relatively limited subpopulations of cells havingsignificantly elevated levels of functional usherin protein can bebeneficial in various patients because in some situations normalizedcells will have a selective advantage relative to diseased cells.However, even modest levels of progenitors with elevated levels offunctional usherin protein can be beneficial for ameliorating one ormore aspects of Usher Syndrome Type 2A in patients. In some examples,about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 90% or more of the photoreceptor cells or retinalprogenitor cells in patients to whom such cells are administered areproducing increased levels of functional usherin protein.

“Administered” refers to the delivery of a progenitor cell compositioninto a subject by a method or route that results in at least partiallocalization of the cell composition at a desired site. A cellcomposition can be administered by any appropriate route that results ineffective treatment in the subject, i.e., administration results indelivery to a desired location in the subject where at least a portionof the composition delivered, i.e. at least 1×10⁴ cells are delivered tothe desired site for a period of time.

In one aspect of the method, the pharmaceutical composition can beadministered via a route such as, but not limited to, enteral (into theintestine), gastroenteral, epidural (into the dura matter), oral (by wayof the mouth), transdermal, peridural, intracerebral (into thecerebrum), intracerebroventricular (into the cerebral ventricles),epicutaneous (application onto the skin), intradermal, (into the skinitself), subcutaneous (under the skin), nasal administration (throughthe nose), intravenous (into a vein), intravenous bolus, intravenousdrip, intraarterial (into an artery), intramuscular (into a muscle),intracardiac (into the heart), intraosseous infusion (into the bonemarrow), intrathecal (into the spinal canal), intraperitoneal, (infusionor injection into the peritoneum), intravesical infusion, intravitreal,(through the eye), intracavernous injection (into a pathologic cavity)intracavitary (into the base of the penis), intravaginal administration,intrauterine, extra-amniotic administration, transdermal (diffusionthrough the intact skin for systemic distribution), transmucosal(diffusion through a mucous membrane), transvaginal, insufflation(snorting), sublingual, sublabial, enema, eye drops (onto theconjunctiva), in ear drops, auricular (in or by way of the ear), buccal(directed toward the cheek), conjunctival, cutaneous, dental (to a toothor teeth), electro-osmosis, endocervical, endosinusial, endotracheal,extracorporeal, hemodialysis, infiltration, interstitial,intra-abdominal, intra-amniotic, intra-articular, intrabiliary,intrabronchial, intrabursal, intracartilaginous (within a cartilage),intracaudal (within the cauda equine), intracisternal (within thecisterna magna cerebellomedularis), intracorneal (within the cornea),dental intracornal, intracoronary (within the coronary arteries),intracorporus cavernosum (within the dilatable spaces of the corporuscavernosa of the penis), intradiscal (within a disc), intraductal(within a duct of a gland), intraduodenal (within the duodenum),intradural (within or beneath the dura), intraepidermal (to theepidermis), intraesophageal (to the esophagus), intragastric (within thestomach), intragingival (within the gingivae), intraileal (within thedistal portion of the small intestine), intralesional (within orintroduced directly to a localized lesion), intraluminal (within a lumenof a tube), intralymphatic (within the lymph), intramedullary (withinthe marrow cavity of a bone), intrameningeal (within the meninges),intramyocardial (within the myocardium), intraocular (within the eye),intraovarian (within the ovary), intrapericardial (within thepericardium), intrapleural (within the pleura), intraprostatic (withinthe prostate gland), intrapulmonary (within the lungs or its bronchi),intrasinal (within the nasal or periorbital sinuses), intraspinal(within the vertebral column), intrasynovial (within the synovial cavityof a joint), intratendinous (within a tendon), intratesticular (withinthe testicle), intrathecal (within the cerebrospinal fluid at any levelof the cerebrospinal axis), intrathoracic (within the thorax),intratubular (within the tubules of an organ), intratumor (within atumor), intratympanic (within the aurus media), intravascular (within avessel or vessels), intraventricular (within a ventricle), iontophoresis(by means of electric current where ions of soluble salts migrate intothe tissues of the body), irrigation (to bathe or flush open wounds orbody cavities), laryngeal (directly upon the larynx), nasogastric(through the nose and into the stomach), occlusive dressing technique(topical route administration, which is then covered by a dressing thatoccludes the area), ophthalmic (to the external eye), oropharyngeal(directly to the mouth and pharynx), parenteral, percutaneous,periarticular, peridural, perineural, periodontal, rectal, respiratory(within the respiratory tract by inhaling orally or nasally for local orsystemic effect), retrobulbar (behind the pons or behind the eyeball),intramyocardial (entering the myocardium), soft tissue, subarachnoid,subconjunctival, submucosal, topical, transplacental (through or acrossthe placenta), transtracheal (through the wall of the trachea),transtympanic (across or through the tympanic cavity), ureteral (to theureter), urethral (to the urethra), vaginal, caudal block, diagnostic,nerve block, biliary perfusion, cardiac perfusion, photopheresis andspinal.

Modes of administration include injection, infusion, instillation,and/or ingestion. “Injection” includes, without limitation, intravenous,intramuscular, intra-arterial, intrathecal, intraventricular,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular,subretinal, subarachnoid, intraspinal, intracerebro spinal, andintrasternal injection and infusion. In some examples, the route isintravenous. For the delivery of cells, administration by injection orinfusion can be made. For the delivery of gRNAs, Cas9, and donortemplates, administration can be by injection into the subretinal space,in close proximity to the photoreceptors.

The cells can be administered systemically. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” refer to theadministration of a population of progenitor cells other than directlyinto a target site, tissue, or organ, such that it enters, instead, thesubject's circulatory system and, thus, is subject to metabolism andother like processes.

The efficacy of a treatment comprising a composition for the treatmentof Usher Syndrome Type 2A can be determined by the skilled clinician.However, a treatment is considered “effective treatment,” if any one orall of the signs or symptoms of, as but one example, levels offunctional usherin are altered in a beneficial manner (e.g., increasedby at least 10%), or other clinically accepted symptoms or markers ofdisease are improved or ameliorated. Efficacy can also be measured byfailure of an individual to worsen as assessed by hospitalization orneed for medical interventions (e.g., progression of the disease ishalted or at least slowed). Methods of measuring these indicators areknown to those of skill in the art and/or described herein. Treatmentincludes any treatment of a disease in an individual or an animal (somenon-limiting examples include a human, or a mammal) and includes: (1)inhibiting the disease, e.g., arresting, or slowing the progression ofsymptoms; or (2) relieving the disease, e.g., causing regression ofsymptoms; and (3) preventing or reducing the likelihood of thedevelopment of symptoms.

The treatment according to the present disclosure can ameliorate one ormore symptoms associated with Usher Syndrome Type 2A by increasing,decreasing or altering the amount of functional usherin in theindividual. Signs typically associated with Usher Syndrome Type 2Ainclude for example, hearing loss and an eye disorder called retinitispigmentosa, which causes night-blindness and a loss of peripheral visionthrough the progressive degeneration of the retina. Many people withUsher syndrome also have severe balance problems.

Kits

The present disclosure provides kits for carrying out the methodsdescribed herein. A kit can include one or more of a genome-targetingnucleic acid, a polynucleotide encoding a genome-targeting nucleic acid,a site-directed polypeptide, a polynucleotide encoding a site-directedpolypeptide, and/or any nucleic acid or proteinaceous molecule necessaryto carry out the aspects of the methods described herein, or anycombination thereof.

A kit can comprise: (1) a vector comprising a nucleotide sequenceencoding a genome-targeting nucleic acid, (2) the site-directedpolypeptide or a vector comprising a nucleotide sequence encoding thesite-directed polypeptide, and (3) a reagent for reconstitution and/ordilution of the vector(s) and or polypeptide.

A kit can comprise: (1) a vector comprising (i) a nucleotide sequenceencoding a genome-targeting nucleic acid, and (ii) a nucleotide sequenceencoding the site-directed polypeptide; and (2) a reagent forreconstitution and/or dilution of the vector.

In any of the above kits, the kit can comprise a single-molecule guidegenome-targeting nucleic acid. In any of the above kits, the kit cancomprise a double-molecule genome-targeting nucleic acid. In any of theabove kits, the kit can comprise two or more double-molecule guides orsingle-molecule guides. The kits can comprise a vector that encodes thenucleic acid targeting nucleic acid.

In any of the above kits, the kit can further comprise a polynucleotideto be inserted to effect the desired genetic modification.

Components of a kit can be in separate containers, or combined in asingle container.

Any kit described above can further comprise one or more additionalreagents, where such additional reagents are selected from a buffer, abuffer for introducing a polypeptide or polynucleotide into a cell, awash buffer, a control reagent, a control vector, a control RNApolynucleotide, a reagent for in vitro production of the polypeptidefrom DNA, adaptors for sequencing and the like. A buffer can be astabilization buffer, a reconstituting buffer, a diluting buffer, or thelike. A kit can also comprise one or more components that can be used tofacilitate or enhance the on-target binding or the cleavage of DNA bythe endonuclease, or improve the specificity of targeting.

In addition to the above-mentioned components, a kit can furthercomprise instructions for using the components of the kit to practicethe methods. The instructions for practicing the methods can be recordedon a suitable recording medium. For example, the instructions can beprinted on a substrate, such as paper or plastic, etc. The instructionscan be present in the kits as a package insert, in the labeling of thecontainer of the kit or components thereof (i.e., associated with thepackaging or sub packaging), etc. The instructions can be present as anelectronic storage data file present on a suitable computer readablestorage medium, e.g., CD-ROM, diskette, flash drive, etc. In someinstances, the actual instructions are not present in the kit, but meansfor obtaining the instructions from a remote source (e.g., via theInternet), can be provided. An example of this case is a kit thatcomprises a web address where the instructions can be viewed and/or fromwhich the instructions can be downloaded. As with the instructions, thismeans for obtaining the instructions can be recorded on a suitablesubstrate.

Additional Therapeutic Approaches

Gene editing can be conducted using nucleases engineered to targetspecific sequences. To date there are four major types of nucleases:meganucleases and their derivatives, zinc finger nucleases (ZFNs),transcription activator like effector nucleases (TALENs), andCRISPR-Cas9 nuclease systems. The nuclease platforms vary in difficultyof design, targeting density and mode of action, particularly as thespecificity of ZFNs and TALENs is through protein-DNA interactions,while RNA-DNA interactions primarily guide Cas9. Cas9 cleavage alsorequires an adjacent motif, the PAM, which differs between differentCRISPR systems. Cas9 from Streptococcus pyogenes cleaves using a NGGPAM, CRISPR from Neisseria meningitidis can cleave at sites with PAMsincluding NNNNGATT, NNNNNGTTT and

NNNNGCTT. A number of other Cas9 orthologs target protospacer adjacentto alternative PAMs.

CRISPR endonucleases, such as Cas9, can be used in the methods of thepresent disclosure. However, the teachings described herein, such astherapeutic target sites, could be applied to other forms ofendonucleases, such as ZFNs, TALENs, HEs, or MegaTALs, or usingcombinations of nucleases. However, in order to apply the teachings ofthe present disclosure to such endonucleases, one would need to, amongother things, engineer proteins directed to the specific target sites.

Additional binding domains can be fused to the Cas9 protein to increasespecificity. The target sites of these constructs would map to theidentified gRNA specified site, but would require additional bindingmotifs, such as for a zinc finger domain. In the case of Mega-TAL, ameganuclease can be fused to a TALE DNA-binding domain. The meganucleasedomain can increase specificity and provide the cleavage. Similarly,inactivated or dead Cas9 (dCas9) can be fused to a cleavage domain andrequire the sgRNA/Cas9 target site and adjacent binding site for thefused DNA-binding domain. This likely would require some proteinengineering of the dCas9, in addition to the catalytic inactivation, todecrease binding without the additional binding site.

Zinc Finger Nucleases

Zinc finger nucleases (ZFNs) are modular proteins comprised of anengineered zinc finger DNA binding domain linked to the catalytic domainof the type II endonuclease FokI. Because FokI functions only as adimer, a pair of ZFNs must be engineered to bind to cognate target“half-site” sequences on opposite DNA strands and with precise spacingbetween them to enable the catalytically active FokI dimer to form. Upondimerization of the FokI domain, which itself has no sequencespecificity per se, a DNA double-strand break is generated between theZFN half-sites as the initiating step in genome editing.

The DNA binding domain of each ZFN is typically comprised of 3-6 zincfingers of the abundant Cys2-His2 architecture, with each fingerprimarily recognizing a triplet of nucleotides on one strand of thetarget DNA sequence, although cross-strand interaction with a fourthnucleotide also can be important. Alteration of the amino acids of afinger in positions that make key contacts with the DNA alters thesequence specificity of a given finger. Thus, a four-finger zinc fingerprotein will selectively recognize a 12 bp target sequence, where thetarget sequence is a composite of the triplet preferences contributed byeach finger, although triplet preference can be influenced to varyingdegrees by neighboring fingers. An important aspect of ZFNs is that theycan be readily re-targeted to almost any genomic address simply bymodifying individual fingers, although considerable expertise isrequired to do this well. In most applications of ZFNs, proteins of 4-6fingers are used, recognizing 12-18 bp respectively. Hence, a pair ofZFNs will typically recognize a combined target sequence of 24-36 bp,not including the typical 5-7 bp spacer between half-sites. The bindingsites can be separated further with larger spacers, including 15-17 bp.A target sequence of this length is likely to be unique in the humangenome, assuming repetitive sequences or gene homologs are excludedduring the design process. Nevertheless, the ZFN protein-DNAinteractions are not absolute in their specificity so off-target bindingand cleavage events do occur, either as a heterodimer between the twoZFNs, or as a homodimer of one or the other of the ZFNs. The latterpossibility has been effectively eliminated by engineering thedimerization interface of the FokI domain to create “plus” and “minus”variants, also known as obligate heterodimer variants, which can onlydimerize with each other, and not with themselves. Forcing the obligateheterodimer prevents formation of the homodimer. This has greatlyenhanced specificity of ZFNs, as well as any other nuclease that adoptsthese FokI variants.

A variety of ZFN-based systems have been described in the art,modifications thereof are regularly reported, and numerous referencesdescribe rules and parameters that are used to guide the design of ZFNs;see, e.g., Segal et al., Proc Natl Acad Sci USA 96(6):2758-63 (1999);Dreier B et al., J Mol Biol 303(4):489-502 (2000); Liu Q et al., J BiolChem 277(6):3850-6 (2002); Dreier et al., J Biol Chem 280(42):35588-97(2005); and Dreier et al., J Biol Chem 276(31):29466-78 (2001).

Transcription Activator-Like Effector Nucleases (TALENs)

Transcription Activator-Like Effector Nucleases (TALENs) representanother format of modular nucleases whereby, as with ZFNs, an engineeredDNA binding domain is linked to the FokI nuclease domain, and a pair ofTALENs operates in tandem to achieve targeted DNA cleavage. The majordifference from ZFNs is the nature of the DNA binding domain and theassociated target DNA sequence recognition properties. The TALEN DNAbinding domain derives from TALE proteins, which were originallydescribed in the plant bacterial pathogen Xanthomonas sp. TALEs arecomprised of tandem arrays of 33-35 amino acid repeats, with each repeatrecognizing a single base pair in the target DNA sequence that istypically up to 20 bp in length, giving a total target sequence lengthof up to 40 bp. Nucleotide specificity of each repeat is determined bythe repeat variable diresidue (RVD), which includes just two amino acidsat positions 12 and 13. The bases guanine, adenine, cytosine and thymineare predominantly recognized by the four RVDs: Asn-Asn, Asn-Ile, His-Aspand Asn-Gly, respectively. This constitutes a much simpler recognitioncode than for zinc fingers, and thus represents an advantage over thelatter for nuclease design. Nevertheless, as with ZFNs, the protein-DNAinteractions of TALENs are not absolute in their specificity, and TALENshave also benefited from the use of obligate heterodimer variants of theFokI domain to reduce off-target activity.

Additional variants of the FokI domain have been created that aredeactivated in their catalytic function. If one half of either a TALENor a ZFN pair contains an inactive FokI domain, then only single-strandDNA cleavage (nicking) will occur at the target site, rather than a DSB.The outcome is comparable to the use of CRISPR/Cas9/Cpf1 “nickase”mutants in which one of the Cas9 cleavage domains has been deactivated.DNA nicks can be used to drive genome editing by HDR, but at lowerefficiency than with a DSB. The main benefit is that off-target nicksare quickly and accurately repaired, unlike the DSB, which is prone toNHEJ-mediated mis-repair.

A variety of TALEN-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., Boch, Science326(5959):1509-12 (2009); Mak et al., Science 335(6069):716-9 (2012);and Moscou et al., Science 326(5959):1501 (2009). The use of TALENsbased on the “Golden Gate” platform, or cloning scheme, has beendescribed by multiple groups; see, e.g., Cermak et al., Nucleic AcidsRes 39(12):e82 (2011); Li et al., Nucleic Acids Res. 39(14):6315-25(2011); Weber et al., PLoS One. 6(2): e16765 (2011); Wang et al., JGenet Genomics 41(6):339-47, Epub 2014 May 17 (2014); and Cermak T etal., Methods Mol Biol 1239:133-59 (2015).

Homing Endonucleases

Homing endonucleases (HEs) are sequence-specific endonucleases that havelong recognition sequences (14-44 base pairs) and cleave DNA with highspecificity—often at sites unique in the genome. There are at least sixknown families of HEs as classified by their structure, includingLAGLIDADG (SEQ ID NO: 5271), GIY-YIG, His-Cis box, H-N-H, PD-(D/E)xK,and Vsr-like that are derived from a broad range of hosts, includingeukarya, protists, bacteria, archaea, cyanobacteria and phage. As withZFNs and TALENs, HEs can be used to create a DSB at a target locus asthe initial step in genome editing. In addition, some natural andengineered HEs cut only a single strand of DNA, thereby functioning assite-specific nickases. The large target sequence of HEs and thespecificity that they offer have made them attractive candidates tocreate site-specific DSBs.

A variety of HE-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., the reviews bySteentoft et al., Glycobiology 24(8):663-80 (2014); Belfort andBonocora, Methods Mol Biol. 1123:1-26 (2014); Hafez and Hausner, Genome55(8):553-69 (2012).

MegaTAL/Tev-mTALEN/MegaTev

As further examples of hybrid nucleases, the MegaTAL platform andTev-mTALEN platform use a fusion of TALE DNA binding domains andcatalytically active HEs, taking advantage of both the tunable DNAbinding and specificity of the TALE, as well as the cleavage sequencespecificity of the HE; see, e.g., Boissel et al., NAR 42: 2591-2601(2014); Kleinstiver et al., G3 4:1155-65 (2014); and Boissel andScharenberg, Methods Mol. Biol. 1239: 171-96 (2015).

In a further variation, the MegaTev architecture is the fusion of ameganuclease (Mega) with the nuclease domain derived from the GIY-YIGhoming endonuclease I-TevI (Tev). The two active sites are positioned˜30 bp apart on a DNA substrate and generate two DSBs withnon-compatible cohesive ends; see, e.g., Wolfs et al., NAR 42, 8816-29(2014). It is anticipated that other combinations of existingnuclease-based approaches will evolve and be useful in achieving thetargeted genome modifications described herein.

dCas9-FokI or dCpf1-FokI and Other Nucleases

Combining the structural and functional properties of the nucleaseplatforms described above offers a further approach to genome editingthat can potentially overcome some of the inherent deficiencies. As anexample, the CRISPR genome editing system typically uses a single Cas9endonuclease to create a DSB. The specificity of targeting is driven bya 20 or 24 nucleotide sequence in the guide RNA that undergoesWatson-Crick base-pairing with the target DNA (plus an additional 2bases in the adjacent NAG or NGG PAM sequence in the case of Cas9 fromS. pyogenes). Such a sequence is long enough to be unique in the humangenome, however, the specificity of the RNA/DNA interaction is notabsolute, with significant promiscuity sometimes tolerated, particularlyin the 5′ half of the target sequence, effectively reducing the numberof bases that drive specificity. One solution to this has been tocompletely deactivate the Cas9 or Cpf1 catalytic function—retaining onlythe RNA-guided DNA binding function—and instead fusing a FokI domain tothe deactivated Cas9; see, e.g., Tsai et al., Nature Biotech 32: 569-76(2014); and Guilinger et al., Nature Biotech 32: 577-82 (2014). BecauseFokI must dimerize to become catalytically active, two guide RNAs arerequired to tether two FokI fusions in close proximity to form the dimerand cleave DNA. This essentially doubles the number of bases in thecombined target sites, thereby increasing the stringency of targeting byCRISPR-based systems.

As further example, fusion of the TALE DNA binding domain to acatalytically active HE, such as I-TevI, takes advantage of both thetunable DNA binding and specificity of the TALE, as well as the cleavagesequence specificity of I-TevI, with the expectation that off-targetcleavage can be further reduced.

Methods, Compositions, Therapeutics, and Kits of the Invention

Accordingly, the present disclosure relates in particular to thefollowing non-limiting inventions:

In a first method, Method 1, the present disclosure provides a methodfor editing an USH2A gene in a human cell, the method comprising:introducing into the human cell one or more DNA endonucleases, therebyeffecting one or more SSBs or DSBs within or near the USH2A gene or aDNA sequence encoding a regulatory sequence of the USH2A gene thatresults in a correction thereby creating an edited human cell.

In another method, Method 2, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: introducing into the human cell one or more DNAendonucleases to effect one or more SSBs or DSBs within or near intron40 of the USH2A gene that results in a correction thereby creating anedited human cell.

In another method, Method 3, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation in a human cellas provided in Method 2, wherein the IVS40 mutation is located withinintron 40 of the USH2A gene.

In another method, Method 4, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A, the methodcomprising: editing an USH2A gene containing an IVS40 mutation in a cellof the patient.

In another method, Method 5, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inMethod 4, wherein the editing comprises: introducing into the cell oneor more DNA endonucleases to effect one or more SSBs or DSBs within ornear intron 40 of the USH2A gene that results in a correction andresults in restoration of usherin protein function.

In another method, Method 6, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 4 or 5, wherein the IVS40 mutation is located withinintron 40 of the USH2A gene.

In another method, Method 7, the present disclosure provides a method asprovided in any one of Methods 1-2 or 5, wherein the one or more DNAendonucleases is a Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7,Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3,Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1,Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16,CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease; ahomolog thereof, a recombination of the naturally occurring moleculethereof, codon-optimized thereof, or modified versions thereof, andcombinations thereof.

In another method, Method 8, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inMethod 7, wherein the method comprises introducing into the cell one ormore polynucleotides encoding the one or more DNA endonucleases.

In another method, Method 9, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inMethod 7, wherein the method comprises introducing into the cell one ormore RNAs encoding the one or more DNA endonucleases.

In another method, Method 10, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 8 or 9, wherein the one or more polynucleotides orone or more RNAs is one or more modified polynucleotides or one or moremodified RNAs.

In another method, Method 11, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inMethod 7, wherein the DNA endonuclease is one or more proteins orpolypeptides.

In another method, Method 12, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 1-11, wherein the method further comprises:introducing into the cell one or more gRNAs.

In another method, Method 13, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inMethod 12, wherein the one or more gRNAs are sgRNAs.

In another method, Method 14, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 12-13, wherein the one or more gRNAs or one or moresgRNAs is one or more modified gRNAs or one or more modified sgRNAs.

In another method, Method 15, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 11-13, wherein the one or more DNA endonucleases ispre-complexed with one or more gRNAs or one or more sgRNAs.

In another method, Method 16, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 1-15, further comprising: introducing into the cell apolynucleotide donor template comprising at least a portion of thewild-type USH2A gene, or cDNA.

In another method, Method 17, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inMethod 16, wherein the at least a portion of the wild-type USH2A gene orcDNA is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8,exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16,exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24,exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32,exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40,exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48,exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56,exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64,exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72,intronic regions, fragments or combinations thereof, or the entire USH2Agene or cDNA.

In another method, Method 18, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 16-17, wherein the donor template is either a singleor double-stranded polynucleotide.

In another method, Method 19, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 16-17, wherein the donor template has homologous armsto the 1q41 region.

In another method, Method 20, the present disclosure provides a methodas provided in any one of Methods 2 or 5, further comprising:introducing into the cell one gRNA; wherein the one or more DNAendonucleases is one or more Cas9 or Cpf1 endonucleases that effect oneSSB or DSB at a locus located within or near intron 40 of the USH2Agene; and wherein the gRNA comprises a spacer sequence that iscomplementary to a segment of the locus located within intron 40.

In another method, Method 21, the present disclosure provides a methodas provided in any one of Methods 2 or 5, further comprising:introducing into the cell one or more gRNAs; wherein the one or more DNAendonucleases is one or more Cas9 or Cpf1 endonucleases that effect apair of SSBs or DSBs, the first at a 5′ locus and the second at a 3′locus, within or near intron 40 of the USH2A gene; and wherein the firstguide RNA comprises a spacer sequence that is complementary to a segmentof the 5′ locus and the second guide RNA comprises a spacer sequencethat is complementary to a segment of the 3′ locus.

In another method, Method 22, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 20 or 21, wherein the IVS40 mutation is locatedwithin intron 40 of the USH2A gene.

In another method, Method 23, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 20-22, wherein the one or more gRNAs are one or moresgRNAs.

In another method, Method 24, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 20-23, wherein the one or more gRNAs or one or moresgRNAs is one or more modified gRNAs or one or more modified sgRNAs.

In another method, Method 25, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 20-24, wherein the one or more DNA endonucleases ispre-complexed with one or more gRNAs or one or more sgRNAs.

In another method, Method 26, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inMethod 20, further comprising: a polynucleotide donor templatecomprising at least a portion of the wild-type USH2A gene; wherein a newsequence from the polynucleotide donor template is inserted into thechromosomal DNA at the locus located within or near intron 40 of theUSH2A gene that results in a correction of the IVS40 mutation in theUSH2A gene.

In another method, Method 27, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inMethod 21, further comprising: a polynucleotide donor templatecomprising at least a portion of the wild-type USH2A gene; wherein a newsequence from the polynucleotide donor template is inserted into thechromosomal DNA between the 5′ locus and the 3′ locus, within or nearintron 40 of the USH2A gene that results in a correction of thechromosomal DNA between the 5′ locus and the 3′ locus within or nearintron 40 of the USH2A gene.

In another method, Method 28, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 20-27, wherein the at least a portion of thewild-type USH2A gene or cDNA is exon 1, exon 2, exon 3, exon 4, exon 5,exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon70, exon 71, exon 72, intronic regions, fragments or combinationsthereof, or the entire USH2A gene or cDNA.

In another method, Method 29, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 20-28, wherein the polynucleotide donor template iseither a single or double-stranded polynucleotide.

In another method, Method 30, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 20-29, wherein the polynucleotide donor template hashomologous arms to the 1q41 region.

In another method, Method 31, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 20-30, wherein the SSB or DSB is located withinintron 40, 0-1800 nucleotides upstream of the IVS40 mutation.

In another method, Method 32, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 20-30, wherein the SSB or DSB is located withinintron 40, 0-1100 nucleotides downstream of the IVS40 mutation.

In another method, Method 33, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 12-15 or 23-25, wherein the gRNA or sgRNA iscomplementary to a segment of intron 40 of the USH2A gene.

In another method, Method 34, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 1-3 or 5-33, wherein the correction is by HDR.

In another method, Method 35, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 26-27, wherein the donor template has homologous armsto the IVS40 mutation.

In another method, Method 36, the present disclosure provides a methodas provided in any one of Methods 2 or 5, further comprising:introducing into the cell two gRNAs; wherein the one or more DNAendonucleases is one or more Cas9 or Cpf1 endonucleases that effect apair of double-strand breaks (DSBs), the first at a 5′ DSB locus and thesecond at a 3′ DSB locus, within or near intron 40 of the USH2A genethat causes a deletion of the chromosomal DNA between the 5′ DSB locusand the 3′ DSB locus that results in a deletion of the chromosomal DNAbetween the 5′ DSB locus and the 3′ DSB locus within or near intron 40of the USH2A gene; and wherein the first guide RNA comprises a spacersequence that is complementary to a segment of the 5′ DSB locus and thesecond guide RNA comprises a spacer sequence that is complementary to asegment of the 3′ DSB locus.

In another method, Method 37, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inMethod 36, wherein the IVS40 mutation is located within intron 40 of theUSH2A gene.

In another method, Method 38, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 36 or 37, wherein the two gRNAs are two sgRNAs.

In another method, Method 39, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 36-38, wherein the two gRNAs or two sgRNAs are twomodified gRNAs or two modified sgRNAs.

In another method, Method 40, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 36-39, wherein the one or more DNA endonucleases ispre-complexed with two gRNAs or two sgRNAs.

In another method, Method 41, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 36-40, wherein the 5′ DSB within intron 40 is located0-1800 nucleotides upstream of the IVS40 mutation.

In another method, Method 42, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 36-40, wherein 3′ DSB within intron 40 is located0-1100 nucleotides downstream of the IVS40 mutation.

In another method, Method 43, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 36-42, wherein the deletion is a deletion of 50 bp to2900 bp.

In another method, Method 44, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 36-42, wherein the deletion is a deletion of 50 bp to2000 bp.

In another method, Method 45, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 36-42, wherein the deletion is a deletion of 50 bp to1000 bp.

In another method, Method 46, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 36-42, wherein the deletion is a deletion of 50 bp to500 bp.

In another method, Method 47, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 36-42, wherein the deletion is a deletion of 50 bp to250 bp.

In another method, Method 48, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 36-47, further comprising: a polynucleotide donortemplate comprising at least a portion of the wild-type USH2A gene.

In another method, Method 49, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 20-22 and 36-37, wherein the Cas9 or Cpf1 mRNA, gRNA,and donor template are either each formulated into separate lipidnanoparticles or all co-formulated into a lipid nanoparticle.

In another method, Method 50, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 20-22 and 36-37, wherein the Cas9 or Cpf1 mRNA, gRNA,and donor template are either each formulated into separate AAV vectorsor all co-formulated into an AAV vector.

In another method, Method 51, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 20-22 and 36-37, wherein the Cas9 or Cpf1 mRNA isformulated into a lipid nanoparticle, and both the gRNA and donortemplate are delivered to the cell by an AAV vector.

In another method, Method 52, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 20-22 and 36-37, wherein the Cas9 or Cpf1 mRNA isformulated into a lipid nanoparticle, and the gRNA is delivered to thecell by electroporation and donor template is delivered to the cell byan AAV vector.

In another method, Method 53, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 50-52, wherein the AAV vector is a self-inactivatingAAV vector.

In another method, Method 54, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inany one of Methods 1-53, wherein the USH2A gene is located on Chromosome1: 215,622,893-216,423,395 (Genome Reference Consortium—GRCh38/hg38).

In another method, Method 55, the present disclosure provides a methodas provided in any one of Methods 1-3 or 5-54, wherein the restorationof usherin protein function is compared to wild-type or normal usherinprotein function.

In another method, Method 56, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inMethod 16, wherein the polynucleotide donor template is up to 11 kb.

In another method, Method 57, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inMethod 56, wherein the polynucleotide donor template is delivered byAAV.

In another method, Method 58, the present disclosure provides a methodas provided in any one of Methods 1-3, wherein the human cell is aphotoreceptor cell or retinal progenitor cell.

In another method, Method 59, the present disclosure provides an in vivomethod for treating a patient with Usher Syndrome Type 2A as provided inMethods 4-57, wherein the cell is a photoreceptor cell or retinalprogenitor cell.

In another method, Method 60, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: editing the USH2A gene containing the IVS40 mutation using agRNA or sgRNA comprising SEQ ID NO: 5321.

In another method, Method 61, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: editing the USH2A gene containing the IVS40 mutation using agRNA or sgRNA comprising SEQ ID NO: 5323.

In another method, Method 62, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: editing the USH2A gene containing the IVS40 mutation using agRNA or sgRNA comprising SEQ ID NO: 5325.

In another method, Method 63, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: editing the USH2A gene containing the IVS40 mutation using agRNA or sgRNA comprising SEQ ID NO: 5327.

In another method, Method 64, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: editing the USH2A gene containing the IVS40 mutation using agRNA or sgRNA comprising SEQ ID NO: 5328.

In another method, Method 65, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: editing the USH2A gene containing the IVS40 mutation using agRNA or sgRNA comprising SEQ ID NO: 5321 and any one of SEQ ID NOs:5267-5269.

In another method, Method 66, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: editing the USH2A gene containing the IVS40 mutation using agRNA or sgRNA comprising SEQ ID NO: 5323 and any one of SEQ ID NOs:5267-5269.

In another method, Method 67, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: editing the USH2A gene containing the IVS40 mutation using agRNA or sgRNA comprising SEQ ID NO: 5325 and any one of SEQ ID NOs:5267-5269.

In another method, Method 68, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: editing the USH2A gene containing the IVS40 mutation using agRNA or sgRNA comprising SEQ ID NO: 5327 and any one of SEQ ID NOs:5267-5269.

In another method, Method 69, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: editing the USH2A gene containing the IVS40 mutation using agRNA or sgRNA comprising SEQ ID NO: 5328 and any one of SEQ ID NOs:5267-5269.

In another method, Method 70, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a gRNA or sgRNA to the patient,wherein the gRNA or sgRNA comprises SEQ ID NO: 5321.

In another method, Method 71, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a gRNA or sgRNA to the patient,wherein the gRNA or sgRNA comprises SEQ ID NO: 5323.

In another method, Method 72, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a gRNA or sgRNA to the patient,wherein the gRNA or sgRNA comprises SEQ ID NO: 5325.

In another method, Method 73, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a gRNA or sgRNA to the patient,wherein the gRNA or sgRNA comprises SEQ ID NO: 5327.

In another method, Method 74, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a gRNA or sgRNA to the patient,wherein the gRNA or sgRNA comprises SEQ ID NO: 5328.

In another method, Method 75, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a gRNA or sgRNA to the patient,wherein the gRNA or sgRNA comprises SEQ ID NO: 5321 and any one of SEQID NOs: 5267-5269.

In another method, Method 76, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a gRNA or sgRNA to the patient,wherein the gRNA or sgRNA comprises SEQ ID NO: 5323 and any one of SEQID NOs: 5267-5269.

In another method, Method 77, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a gRNA or sgRNA to the patient,wherein the gRNA or sgRNA comprises SEQ ID NO: 5325 and any one of SEQID NOs: 5267-5269.

In another method, Method 78, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a gRNA or sgRNA to the patient,wherein the gRNA or sgRNA comprises SEQ ID NO: 5327 and any one of SEQID NOs: 5267-5269.

In another method, Method 79, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a gRNA or sgRNA to the patient,wherein the gRNA or sgRNA comprises SEQ ID NO: 5328 and any one of SEQID NOs: 5267-5269.

In another method, Method 80, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: deleting a sequence comprising the IVS40 mutation using afirst gRNA or sgRNA comprising SEQ ID NO: 5295 and a second gRNA orsgRNA comprising SEQ ID NO: 5279.

In another method, Method 81, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: deleting a sequence comprising the IVS40 mutation using afirst gRNA or sgRNA comprising SEQ ID NO: 5294 and a second gRNA orsgRNA comprising SEQ ID NO: 5300.

In another method, Method 82, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: deleting a sequence comprising the IVS40 mutation using afirst gRNA or sgRNA comprising SEQ ID NO: 5295 and a second gRNA orsgRNA comprising SEQ ID NO: 5300.

In another method, Method 83, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: deleting a sequence comprising the IVS40 mutation using afirst gRNA or sgRNA comprising SEQ ID NO: 5290 and a second gRNA orsgRNA comprising SEQ ID NO: 5300.

In another method, Method 84, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: deleting a sequence comprising the IVS40 mutation using afirst gRNA or sgRNA comprising SEQ ID NO: 5277 and a second gRNA orsgRNA comprising SEQ ID NO: 5300.

In another method, Method 85, the present disclosure provides a methodof any one of Methods 80-84, wherein the first gRNA or sgRNA and secondgRNA or sgRNA are administered simultaneously; the first gRNA or sgRNAis administered prior to the second gRNA or sgRNA; or the second gRNA orsgRNA is administered prior to the first gRNA or sgRNA.

In another method, Method 86, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a first gRNA or sgRNA and secondgRNA or sgRNA to the patient, wherein the first gRNA or sgRNA comprisesSEQ ID NO: 5295 and the second gRNA or sgRNA comprises SEQ ID NO: 5279.

In another method, Method 87, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a first gRNA or sgRNA and secondgRNA or sgRNA to the patient, wherein the first gRNA or sgRNA comprisesSEQ ID NO: 5294 and the second gRNA or sgRNA comprises SEQ ID NO: 5300.

In another method, Method 88, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a first gRNA or sgRNA and secondgRNA or sgRNA to the patient, wherein the first gRNA or sgRNA comprisesSEQ ID NO: 5295 and the second gRNA or sgRNA comprises SEQ ID NO: 5300.

In another method, Method 89, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a first gRNA or sgRNA and secondgRNA or sgRNA to the patient, wherein the first gRNA or sgRNA comprisesSEQ ID NO: 5290 and the second gRNA or sgRNA comprises SEQ ID NO: 5300.

In another method, Method 90, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a first gRNA or sgRNA and secondgRNA or sgRNA to the patient, wherein the first gRNA or sgRNA comprisesSEQ ID NO: 5277 and the second gRNA or sgRNA comprises SEQ ID NO: 5300.

In another method, Method 91, the present disclosure provides a methodof any one of Methods 86-90, wherein the first gRNA or sgRNA and secondgRNA or sgRNA are administered simultaneously; the first gRNA or sgRNAis administered prior to the second gRNA or sgRNA; or the second gRNA orsgRNA is administered prior to the first gRNA or sgRNA to the patient.

In another method, Method 92, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: deleting a sequence comprising the IVS40 mutation using afirst gRNA or sgRNA comprising SEQ ID NO: 5452 and a second gRNA orsgRNA comprising SEQ ID NO: 5449.

In another method, Method 93, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: deleting a sequence comprising the IVS40 mutation using afirst gRNA or sgRNA comprising SEQ ID NO: 5453 and a second gRNA orsgRNA comprising SEQ ID NO: 5449.

In another method, Method 94, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: deleting a sequence comprising the IVS40 mutation using afirst gRNA or sgRNA comprising SEQ ID NO: 5455 and a second gRNA orsgRNA comprising SEQ ID NO: 5457.

In another method, Method 95, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: deleting a sequence comprising the IVS40 mutation using afirst gRNA or sgRNA comprising SEQ ID NO: 5452 and a second gRNA orsgRNA comprising SEQ ID NO: 5451.

In another method, Method 96, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation, the methodcomprising: deleting a sequence comprising the IVS40 mutation using afirst gRNA or sgRNA comprising SEQ ID NO: 5448 and a second gRNA orsgRNA comprising SEQ ID NO: 5449.

In another method, Method 97, the present disclosure provides a methodof any one of Methods 92-96, wherein the first gRNA or sgRNA and secondgRNA or sgRNA are administered simultaneously; the first gRNA or sgRNAis administered prior to the second gRNA or sgRNA; or the second gRNA orsgRNA is administered prior to the first gRNA or sgRNA.

In another method, Method 98, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a first gRNA or sgRNA and secondgRNA or sgRNA to the patient, wherein the first gRNA or sgRNA comprisesSEQ ID NO: 5452 and the second gRNA or sgRNA comprises SEQ ID NO: 5449.

In another method, Method 99, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a first gRNA or sgRNA and secondgRNA or sgRNA to the patient, wherein the first gRNA or sgRNA comprisesSEQ ID NO: 5453 and the second gRNA or sgRNA comprises SEQ ID NO: 5449.

In another method, Method 100, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a first gRNA or sgRNA and secondgRNA or sgRNA to the patient, wherein the first gRNA or sgRNA comprisesSEQ ID NO: 5455 and the second gRNA or sgRNA comprises SEQ ID NO: 5457.

In another method, Method 101, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a first gRNA or sgRNA and secondgRNA or sgRNA to the patient, the first gRNA or sgRNA comprises SEQ IDNO: 5452 and the second gRNA or sgRNA comprises SEQ ID NO: 5451.

In another method, Method 102, the present disclosure provides a methodfor treating a patient with an USH2A gene containing an IVS40 mutation,the method comprising: administering a first gRNA or sgRNA and secondgRNA or sgRNA to the patient, wherein the first gRNA or sgRNA comprisesSEQ ID NO: 5448 and the second gRNA or sgRNA comprises SEQ ID NO: 5449.

In another method, Method 103, the present disclosure provides a methodof any one of Methods 98-102, wherein the first gRNA or sgRNA and secondgRNA or sgRNA are administered simultaneously; the first gRNA or sgRNAis administered prior to the second gRNA or sgRNA; or the second gRNA orsgRNA is administered prior to the first gRNA or sgRNA.

In another method, Method 104, the present disclosure provides a methodfor editing a USH2A gene in a human cell as provided in Method 1,wherein the human cell has defective activity and the edited human cellexpresses a functional USH2A.

In another method, Method 105, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation in a human cellas provided in Method 2, wherein the human cell has defective activityand the edited human cell expresses a functional USH2A.

In another method, Method 106, the present disclosure provides a methodfor editing a USH2A gene in a human cell as provided in Method 1,wherein the correction results in a modulation of expression or functionof the USH2A gene.

In another method, Method 107, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation in a human cellas provided in Method 2, wherein the correction results in a modulationof expression or function of the USH2A gene.

In another method, Method 108, the present disclosure provides an invivo method for treating a patient with Usher Syndrome Type 2A asprovided in Method 5, wherein the correction results in a modulation ofexpression or function of the USH2A gene and results in restoration ofusherin protein function.

In another method, Method 109, the present disclosure provides an invivo method for treating a patient with Usher Syndrome Type 2A asprovided in Method 4, wherein the editing comprises: introducing intothe cell one or more DNA endonucleases to effect one or more SSBs orDSBs within or near intron 40 of the USH2A gene that results in amodulation of expression or function of the USH2A gene and results inrestoration of usherin protein function.

In another method, Method 110, the present disclosure provides a methodfor editing a USH2A gene in a human cell, the method comprising:introducing into the human cell one or more DNA endonucleases to effectone or more SSBs or DSBs within or near the USH2A gene or other DNAsequences that encode regulatory sequence of the USH2A gene that resultsin a modulation of expression or function of the USH2A gene therebycreating an edited human cell.

In another method, Method 111, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation in a human cell,the method comprising: introducing into the human cell one or more DNAendonucleases to effect one or more SSBs or DSBs within or near intron40 of the USH2A gene that results in a modulation of expression orfunction of the USH2A gene thereby creating an edited human cell.

In another method, Method 112, the present disclosure provides a methodfor editing an USH2A gene containing an IVS40 mutation in a human cellas provided in Method 111, wherein the IVS40 mutation is located withinintron 40 of the USH2A gene.

In a first composition, Composition 1, the present disclosure providesone or more gRNAs for editing an IVS40 mutation in a USH2A gene in acell from a patient with Usher Syndrome Type 2A, the one or more gRNAscomprising a spacer sequence selected from the group consisting ofnucleic acid sequences in SEQ ID NOs: 5272-5319, 5321, 5323, 5325,5327-5328, 5443, and 5446-5461 of the Sequence Listing.

In another composition, Composition 2, the present disclosure providesone or more gRNAs of Composition 1, wherein the IVS40 mutation islocated within intron 40 of the USH2A gene.

In another composition, Composition 3, the present disclosure providesone or more gRNAs of any of Compositions 1 or 2, wherein the one or moregRNAs are one or more sgRNAs.

In another composition, Composition 4, the present disclosure providesone or more gRNAs of any of Compositions 1-3, wherein the one or moregRNAs or one or more sgRNAs is one or more modified gRNAs or one or moremodified sgRNAs.

In another composition, Composition 5, the present disclosure providesone or more gRNAs of any of Compositions 1-4, wherein the cell is aphotoreceptor cell, retinal progenitor cell, mesenchymal stem cell(MSC), or induced pluripotent stem cell (iPSC).

In another composition, Composition 6, the present disclosure provides asingle-molecule guide RNA (sgRNA) for editing an IVS40 mutation in aUSH2A gene in a cell from a patient with Usher Syndrome Type 2A, thesgRNA comprising SEQ ID NO: 5321.

In another composition, Composition 7, the present disclosure provides asingle-molecule guide RNA (sgRNA) for editing an IVS40 mutation in aUSH2A gene in a cell from a patient with Usher Syndrome Type 2A, thesgRNA comprising SEQ ID NO: 5323.

In another composition, Composition 8, the present disclosure provides asingle-molecule guide RNA (sgRNA) for editing an IVS40 mutation in aUSH2A gene in a cell from a patient with Usher Syndrome Type 2A, thesgRNA comprising SEQ ID NO: 5325.

In another composition, Composition 9, the present disclosure provides asingle-molecule guide RNA (sgRNA) for editing an IVS40 mutation in aUSH2A gene in a cell from a patient with Usher Syndrome Type 2A, thesgRNA comprising SEQ ID NO: 5327.

In another composition, Composition 10, the present disclosure providesa single-molecule guide RNA (sgRNA) for editing an IVS40 mutation in aUSH2A gene in a cell from a patient with Usher Syndrome Type 2A, thesgRNA comprising SEQ ID NO: 5328.

In another composition, Composition 11, the present disclosure providesa single-molecule guide RNA (sgRNA) for editing an IVS40 mutation in aUSH2A gene in a cell from a patient with Usher Syndrome Type 2A, thesgRNA comprising SEQ ID NO: 5321 and any one of SEQ ID NOs: 5267-5269.

In another composition, Composition 12, the present disclosure providesa single-molecule guide RNA (sgRNA) for editing an IVS40 mutation in aUSH2A gene in a cell from a patient with Usher Syndrome Type 2A, thesgRNA comprising SEQ ID NO: 5323 and any one of SEQ ID NOs: 5267-5269.

In another composition, Composition 13, the present disclosure providesa single-molecule guide RNA (sgRNA) for editing an IVS40 mutation in aUSH2A gene in a cell from a patient with Usher Syndrome Type 2A, thesgRNA comprising SEQ ID NO: 5325 and any one of SEQ ID NOs: 5267-5269.

In another composition, Composition 14, the present disclosure providesa single-molecule guide RNA (sgRNA) for editing an IVS40 mutation in aUSH2A gene in a cell from a patient with Usher Syndrome Type 2A, thesgRNA comprising SEQ ID NO: 5327 and any one of SEQ ID NOs: 5267-5269.

In another composition, Composition 15, the present disclosure providesa single-molecule guide RNA (sgRNA) for editing an IVS40 mutation in aUSH2A gene in a cell from a patient with Usher Syndrome Type 2A, thesgRNA comprising SEQ ID NO: 5328 and any one of SEQ ID NOs: 5267-5269.

In another composition, Composition 16, the present disclosure providesone or more gRNAs for editing an IVS40 mutation in a USH2A gene, the oneor more gRNAs comprising a spacer sequence selected from the groupconsisting of nucleic acid sequences in SEQ ID NOs: 5272-5319, 5321,5323, 5325, 5327-5328, 5443, and 5446-5461 of the Sequence Listing.

In a first therapeutic, Therapeutic 1, the present disclosure provides atherapeutic for treating a patient with Usher Syndrome Type 2A, thetherapeutic comprising at least one or more gRNAs for editing an IVS40mutation in a USH2A gene, the one or more gRNAs comprising a spacersequence selected from the group consisting of nucleic acid sequences inSEQ ID NOs: 5272-5319, 5321, 5323, 5325, 5327-5328, 5443, and 5446-5461of the Sequence Listing.

In another therapeutic, Therapeutic 2, the present disclosure providesthe therapeutic of Therapeutic 1, wherein the IVS40 mutation is locatedwithin intron 40 of the USH2A gene.

In another therapeutic, Therapeutic 3, the present disclosure providesthe therapeutic of any one of Therapeutics 1 or 2, wherein the one ormore gRNAs are one or more sgRNAs.

In another therapeutic, Therapeutic 4, the present disclosure providesthe therapeutic of any one of Therapeutics 1-3, wherein the one or moregRNAs or one or more sgRNAs is one or more modified gRNAs or one or moremodified sgRNAs.

In another therapeutic, Therapeutic 5, the present disclosure provides atherapeutic for treating a patient with Usher Syndrome Type 2A, thetherapeutic formed by a method comprising: introducing one or more DNAendonucleases; introducing one or more gRNA or one or more sgRNA forediting an IVS40 mutation in a USH2A gene; optionally introducing one ormore donor template; wherein the one or more gRNAs or sgRNAs comprise aspacer sequence selected from the group consisting of nucleic acidsequences in SEQ ID NOs: 5272-5319, 5321, 5323, 5325, 5327-5328, 5443,and 5446-5461 of the Sequence Listing.

In another therapeutic, Therapeutic 6, the present disclosure providesthe therapeutic of Therapeutic 5, wherein the IVS40 mutation is locatedwithin intron 40 of the USH2A gene.

In a first kit, Kit 1, the present disclosure provides a kit fortreating a patient with Usher Syndrome Type 2A in vivo, the kitcomprising one or more gRNAs or sgRNAs for editing an IVS40 mutation ina USH2A gene wherein the one or more gRNAs or sgRNAs comprise a spacersequence selected from the group consisting of nucleic acid sequences inSEQ ID NOs: 5272-5319, 5321, 5323, 5325, 5327-5328, 5443, and 5446-5461of the Sequence Listing; one or more DNA endonucleases; and optionally,one or more donor template.

In another kit, Kit 2, the present disclosure provides the kit of Kit 1,wherein the IVS40 mutation is located within intron 40 of the USH2Agene.

In another kit, Kit 3, the present disclosure provides the kit of anyone of Kits 1 or 2, wherein the one or more DNA endonucleases is a Cas1,Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known asCsn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5,Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1,Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1,Csf2, Csf3, Csf4, or Cpf1 endonuclease; a homolog thereof, arecombination of the naturally occurring molecule thereof,codon-optimized thereof, or modified versions thereof, and combinationsthereof.

In another kit, Kit 4, the present disclosure provides the kit of anyone of Kits 1-3, comprising one or more donor template.

In another kit, Kit 5, the present disclosure provides the kit of Kit 4,wherein the donor template has homologous arms to the 1q41 region.

In another kit, Kit 6, the present disclosure provides the kit of Kit 4,wherein the donor template has homologous arms to the IVS40 mutation.

In a first nucleic acid, Nucleic Acid 1, the present disclosure providesa nucleic acid encoding a gRNA comprising a spacer sequence selectedfrom the group consisting of SEQ ID NOs: 5272-5319, 5321, 5323, 5325,5327-5328, 5443, and 5446-5461.

In another nucleic acid, Nucleic Acid 2, the present disclosure providesthe nucleic acid of Nucleic Acid 1, wherein the gRNA is a sgRNA.

In a first vector, Vector 1, the present disclosure provides a vectorencoding a gRNA comprising a spacer sequence selected from the groupconsisting of SEQ ID NOs: 5272-5319, 5321, 5323, 5325, 5327-5328, 5443,and 5446-5461.

In another vector, Vector 2, the present disclosure provides the vectorof Vector 1, wherein the gRNA is a sgRNA.

In another vector, Vector 3, the present disclosure provides the vectorof any one of Vectors 1 or 2, wherein the vector is an AAV.

In another vector, Vector 4, the present disclosure provides the vectorof any one of Vectors 1-3, wherein the vector is an AAV5 serotype capsidvector.

Definitions

In addition to the definitions previously set forth herein, thefollowing definitions are relevant to the present disclosure:

As used herein, the term “gene” refers to a segment of nucleic acidwhich encodes and is capable of expressing a specific gene product. Agene often produces a protein or polypeptide as its gene product, but agene can produce any desired polypeptide or nucleic acid product.

The term “alteration” or “alteration of genetic information” refers toany change in the genome of a cell.

The term “insertion” refers to an addition of one or more nucleotides ina DNA sequence. Insertions can range from small insertions of a fewnucleotides to insertions of large segments such as a cDNA or a gene.

The term “deletion” refers to a loss or removal of one or morenucleotides in a DNA sequence or a loss or removal of the function of agene. In some cases, a deletion can include, for example, a loss of afew nucleotides, an exon, an intron, a gene segment, or the entiresequence of a gene. In some cases, deletion of a gene refers to theelimination or reduction of the function or expression of a gene or itsgene product. This can result from not only a deletion of sequenceswithin or near the gene, but also other events (e.g., insertion,nonsense mutation) that disrupt the expression of the gene.

The term “correction” as used herein, refers to a change of one or morenucleotides of a genome in a cell. Non-limiting examples of a changeinclude an insertion, deletion, substitution, integration, inversion, orduplication. Such correction may result in a more favorable genotypic orphenotypic outcome, whether in structure or function, for the genomicsite which is corrected. One non-limiting example of a “correction”includes the change of a mutant or defective sequence which restoresstructure or function to a gene or its gene product(s). Depending on thenature of the mutation, correction may be achieved via variousstrategies disclosed herein.

By “hybridizable” or “complementary” or “substantially complementary” itis meant that a nucleic acid (e.g., RNA) comprises a sequence ofnucleotides that enables it to non-covalently bind, e.g.: formWatson-Crick base pairs, “anneal”, or “hybridize,” to another nucleicacid in a sequence-specific, antiparallel manner (i.e., a nucleic acidspecifically binds to a complementary nucleic acid) under theappropriate in vitro and/or in vivo conditions of temperature andsolution ionic strength. As is known in the art, standard Watson-Crickbase-pairing includes: adenine (A) pairing with thymidine (T), adenine(A) pairing with uracil (U), and guanine (G) pairing with cytosine (C)[DNA, RNA]. In some examples, a spacer sequence of a gRNA or sgRNA maybe fully complementary to a nucleotide sequence. In other examples, aspacer sequence of a gRNA or sgRNA may be fully complementary to anucleotide sequence except for in at least one location. In otherexamples, a spacer sequence of a gRNA or sgRNA may be fullycomplementary to the nucleotide sequence except for in at least twolocations.

The term “knock-in” refers to an addition of a DNA sequence, or fragmentthereof into a genome. Such DNA sequences to be knocked-in may includean entire gene or genes, may include regulatory sequences associatedwith a gene or any portion or fragment of the foregoing. For example, acDNA encoding the wild-type protein may be inserted into the genome of acell carrying a mutant gene. Knock-in strategies need not replace thedefective gene, in whole or in part. In some cases, a knock-in strategymay further involve substitution of an existing sequence with theprovided sequence, e.g., substitution of a mutant allele with awild-type copy. On the other hand, the term “knock-out” refers to theelimination of a gene or the expression of a gene. For example, a genecan be knocked out by either a deletion or an addition of a nucleotidesequence that leads to a disruption of the reading frame. As anotherexample, a gene may be knocked out by replacing a part of the gene withan irrelevant sequence. Finally, the term “knock-down” as used hereinrefers to reduction in the expression of a gene or its gene product(s).As a result of a gene knock-down, the protein activity or function maybe attenuated or the protein levels may be reduced or eliminated.

The term “comprising” or “comprises” is used in reference tocompositions, therapeutics, kits, methods, and respective component(s)thereof, that are essential to the present disclosure, yet open to theinclusion of unspecified elements, whether essential or not.

The term “consisting essentially of” refers to those elements requiredfor a given aspect. The term permits the presence of additional elementsthat do not materially affect the basic and novel or functionalcharacteristic(s) of that aspect of the present disclosure.

The term “consisting of” refers to compositions, therapeutics, kits,methods, and respective components thereof as described herein, whichare exclusive of any element not recited in that description of theaspect.

The singular forms “a,” “an,” and “the” include plural references,unless the context clearly dictates otherwise.

Any numerical range recited in this specification describes allsub-ranges of the same numerical precision (i.e., having the same numberof specified digits) subsumed within the recited range. For example, arecited range of “1.0 to 10.0” describes all sub-ranges between (andincluding) the recited minimum value of 1.0 and the recited maximumvalue of 10.0, such as, for example, “2.4 to 7.6,” even if the range of“2.4 to 7.6” is not expressly recited in the text of the specification.Accordingly, the Applicant reserves the right to amend thisspecification, including the claims, to expressly recite any sub-rangeof the same numerical precision subsumed within the ranges expresslyrecited in this specification. All such ranges are inherently describedin this specification such that amending to expressly recite any suchsub-ranges will comply with written description, sufficiency ofdescription, and added matter requirements, including the requirementsunder 35 U.S.C. § 112(a) and Article 123(2) EPC. Also, unless expresslyspecified or otherwise required by context, all numerical parametersdescribed in this specification (such as those expressing values,ranges, amounts, percentages, and the like) may be read as if prefacedby the word “about,” even if the word “about” does not expressly appearbefore a number. Additionally, numerical parameters described in thisspecification should be construed in light of the number of reportedsignificant digits, numerical precision, and by applying ordinaryrounding techniques. It is also understood that numerical parametersdescribed in this specification will necessarily possess the inherentvariability characteristic of the underlying measurement techniques usedto determine the numerical value of the parameter.

Any patent, publication, or other disclosure material identified hereinis incorporated by reference into this specification in its entiretyunless otherwise indicated, but only to the extent that the incorporatedmaterial does not conflict with existing descriptions, definitions,statements, or other disclosure material expressly set forth in thisspecification. As such, and to the extent necessary, the expressdisclosure as set forth in this specification supersedes any conflictingmaterial incorporated by reference. Any material, or portion thereof,that is said to be incorporated by reference into this specification,but which conflicts with existing definitions, statements, or otherdisclosure material set forth herein, is only incorporated to the extentthat no conflict arises between that incorporated material and theexisting disclosure material. Applicants reserve the right to amend thisspecification to expressly recite any subject matter, or portionthereof, incorporated by reference herein.

The details of one or more aspects of the present disclosure are setforth in the accompanying examples below. Although any materials andmethods similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, specific examples ofthe materials and methods contemplated are now described. Otherfeatures, objects and advantages of the present disclosure will beapparent from the description. In the description examples, the singularforms also include the plural unless the context clearly dictatesotherwise. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this present disclosure belongs. Inthe case of conflict, the present description will control.

EXAMPLES

The present disclosure will be more fully understood by reference to thefollowing examples, which provide illustrative non-limiting aspects ofthe invention.

The examples describe the use of the CRISPR system as an illustrativegenome editing technique to create defined therapeutic genomicdeletions, insertions, or replacements within or near the IVS40 mutationin the USH2A gene that lead to a disruption or excision of the IVS40mutation in the genomic locus that restores usherin protein function.Introduction of the defined therapeutic modifications represents a noveltherapeutic strategy for the potential amelioration, if not elimination,of Usher Syndrome Type 2A, as described and illustrated herein.

Example 1 CRISPR/S. pyogenes (Sp)Cas9 PAM Sites for the IVS40 Mutationin the USH2A Gene

To discover target sites for genome editing by SpCas9, a region spanning1800 nucleotides upstream and 1100 nucleotides downstream of the IVS40mutation in intron 40 of the USH2A gene (2.9 kB total) was scanned forSpCas9 protospacer adjacent motifs (PAMs). The area was scanned for PAMshaving the sequence NRG. gRNA spacer sequences (17-24 bps) locatedimmediately upstream of the NRG PAM were then identified. Thesesequences are candidates for use in editing the gene.

Example 2 CRISPR/S. aureus(Sa)Cas9 PAM Sites for the IVS40 Mutation inthe USH2A Gene

To discover target sites for genome editing by SaCas9, a region spanning1100 nucleotides upstream and 550 nucleotides downstream of the IVS40mutation in intron 40 of the USH2A gene was scanned for SaCas9 PAMs. Thearea was scanned for PAMs having the sequence NNGRRT. gRNA spacersequences (17-24 bps) located immediately upstream of the NNGRRT PAMwere then identified. These sequences are candidates for use in editingthe gene.

Example 3 CRISPR/S. thermophilus(St)Cas9 PAM Sites for the IVS40Mutation in the USH2A Gene

To discover target sites for genome editing by StCas9, a region spanning1800 nucleotides upstream and 1100 nucleotides downstream of the IVS40mutation in intron 40 of the USH2A gene (2.9 kB total) is scanned forStCas9 PAMs. The area is scanned for PAMs having the sequence NNAGAAW.gRNA spacer sequences (17-24 bps) located immediately upstream of theNNAGAAW PAM are then identified. These sequences are candidates for usein editing the gene.

Example 4 CRISPR/T. denticola(Td)Cas9 PAM Sites for the IVS40 Mutationin the USH2A Gene

To discover target sites for genome editing by TdCas9, a region spanning1800 nucleotides upstream and 1100 nucleotides downstream of the IVS40mutation in intron 40 of the USH2A gene (2.9 kB total) is scanned forTdCas9 PAMs. The area is scanned for PAMs having the sequence NAAAAC.gRNA spacer sequences (17-24 bps) located immediately upstream of theNAAAAC PAM are then identified. These sequences are candidates for usein editing the gene.

Example 5 CRISPR/N. meningitides (Nm)Cas9 PAM Sites for the IVS40Mutation in the USH2A Gene

To discover target sites for genome editing by NmCas9, a region spanning1800 nucleotides upstream and 1100 nucleotides downstream of the IVS40mutation in intron 40 of the USH2A gene (2.9 kB total) is scanned forNmCas9 PAMs. The area is scanned for PAMs having the sequence NNNNGHTT.gRNA spacer sequences (17-24 bps) located immediately upstream of theNNNNGHTT PAMs are then identified. These sequences are candidates foruse in editing the gene.

Example 6 CRISPR/Cpf1 PAM Sites for the IVS40 Mutation in the USH2A Gene

To discover target sites for genome editing by Cpf-1, a region spanning1800 nucleotides upstream and 1100 nucleotides downstream of the IVS40mutation in intron 40 of the USH2A gene (2.9 kB total) is scanned forCpf1 PAMs. The area is scanned for PAMs having the sequence YTN. gRNAspacer sequences (17-24 bps.) located immediately upstream of the YTNPAM are then identified. These sequences are candidates for use inediting the gene.

Example 7 Bioinformatics Analysis of the Guide RNA Strands

A gRNA or sgRNA can direct an RNP complex to an on-target site such as agenomic sequence for which editing is desired but may also have thepotential to interact with an off-target site for which editing is notdesired. To identify which candidate gRNAs that were likely to haveon-target and/or off-target activity, candidate gRNAs were screened andselected in a single process or multi-step process that used both insilico analysis of binding and experimentally assessed activity at bothon-target and off-target sites.

By way of illustration, candidate gRNAs having sequences that match aparticular on-target site, such as a site within or near the IVS40mutation in the USH2A gene, with an adjacent PAM were assessed for theirpotential to cleave at off-target sites having similar sequences, usingone or more of a variety of bioinformatics tools available for assessingoff-target binding, as described and illustrated in more detail below,in order to assess the likelihood of effects at chromosomal positionsother than those intended.

Candidates predicted to have relatively lower potential for off-targetactivity were then assessed experimentally to measure their on-targetactivity, and then off-target activities at various sites. gRNAs havingsufficiently high on-target activity to achieve desired levels of geneediting at the selected locus, and relatively lower off-target activityto reduce the likelihood of alterations at other chromosomal loci werepreferred. The ratio of on-target to off-target activity is oftenreferred to as the “specificity” of a gRNA.

For initial screening of predicted off-target activities, there were anumber of bioinformatics tools known and publicly available that wereused to predict the most likely off-target sites; and since binding totarget sites in the CRISPR/Cas9/Cpf1 nuclease system is driven byWatson-Crick base pairing between complementary sequences, the degree ofdissimilarity (and therefore reduced potential for off-target binding)was essentially related to primary sequence differences: mismatches andbulges, i.e. bases that were changed to a non-complementary base, andinsertions or deletions of bases in the potential off-target siterelative to the target site. An exemplary bioinformatics tool calledCOSMID (CRISPR Off-target Sites with Mismatches, Insertions andDeletions) (available on the web at crispr.bme.gatech.edu) compiles suchsimilarities. Other bioinformatics tools include, but are not limited toautoCOSMID and CCTop.

Bioinformatic tools were used to minimize off-target cleavage in orderto reduce the detrimental effects of mutations and chromosomalrearrangements. Studies on CRISPR/Cas9 systems suggested the possibilityof off-target activity due to non-specific hybridization of the guidestrand to DNA sequences with base pair mismatches and/or bulges,particularly at positions distal from the PAM region. Therefore, it wasimportant to have a bioinformatics tool that identified potentialoff-target sites that have insertions and/or deletions between the RNAguide strand and genomic sequences, in addition to base-pair mismatches.Bioinformatics tools based upon the off-target prediction algorithmCCTop were used to search genomes for potential CRISPR off-target sites(CCTop is available on the web at crispr.cos.uni-heidelberg.de/). Theoutput ranked lists of the potential off-target sites based on thenumber and location of mismatches, allowing more informed choice oftarget sites, and avoiding the use of sites with more likely off-targetcleavage.

Additional bioinformatics pipelines were employed that weigh theestimated on- and/or off-target activity of gRNA targeting sites in aregion. Other features that were used to predict activity includeinformation about the cell type in question, DNA accessibility,chromatin state, transcription factor binding sites, transcriptionfactor binding data, and other CHIP-seq data. Additional factors wereweighed that predict editing efficiency, such as relative positions anddirections of pairs of gRNAs, local sequence features andmicro-homologies.

These processes allowed for selection of high specificity gRNAs.

Example 8 Generation of an IVS40 Mutant Cell Line

To test candidate gRNAs for on-target activity against genomic DNA, aHEK 293 cell line with a homozygous C.7595-2144A>G mutation (IVS40 USH2Amutant cell line) was generated using wild-type HEK 293 cells. Wild-typeHEK 293 cells contain two wild-type alleles of the human USH2A gene, andendogenously express low levels of USH2A.

The wild-type HEK 293 cells were transfected with ribonucleoproteins(RNPs) and single-stranded DNA oligos (3-6 μg) using Lonza'sNucleofector™ kit, Lonza's nucleofector machine, and the recommendedprogram for HEK 293 cells (available from Lonza, Switzerland).

Ribonucleoproteins (RNPs) were made with 2.5 μg TrueCut™ Cas9 Protein v2(available from ThermoFisher Scientific, Massachusetts, US) and a 1 μg(˜25 pM) synthetic gRNA (from ThermoFisher Scientific). The syntheticgRNA comprised the following unmodified protospacer region:UAAAGAUGAUCUCUUAUCUU (SEQ ID NO: 5326) and

ThermoFisher's proprietary tracrRNA sequence. The synthetic gRNA used tointroduce a double-stranded break in the wild-type HEK 293 genome wasdesigned so that the PAM sequence recognized by the RNP would no longerserve as a PAM sequence once the desired IVS40 mutation was introducedto the genome, thus preventing further editing of the genomes of cellsthat have undergone successful HDR (FIG. 4A).

The single-stranded DNA oligo used as a template for HDR was:GCACTTCAAACCCCCACAATACACAGCCTTTTCTTAAAGATGATCTCTTACCTTGGGAAAGGAGAGGTGTTCAATTTCAATTTCATGATTTGTTTCCCCCT (SEQ ID NO: 5494).

The transfected cells were allowed to recover in culture for 3-7 dayspost-transfection. Single cells were automatically sorted into 96-wellplates and colonies originated from the single cells. Genomic DNA wasisolated from each colony of cells, PCR amplified using a forward primerAGTTGCAGGCCAGTTGATTTGTAT (SEQ ID NO: 5495) and reverse primerCAAAATGGGGATACAGCTCCTTTC (SEQ ID NO: 5496), and the PCR product wassequenced for the presence of the desired C.7595-2144A>G (IVS40) singlenucleotide mutation in the human USH2A gene. Several clones wereisolated that had the IVS40 mutation introduced into both alleles (FIG.4B). Thus, applicants generated a cell line appropriate for use inon-target editing experiments.

Example 9 Testing of sgRNAs (USH2A MO, USH2A MG, USH2A MB, USH2A MP, andUSH2A MR)

For selected gRNAs predicted to have the lowest off-target activity,on-target activity was tested using the IVS40 USH2A mutant cell lineobtained in Example 8.

The IVS40 USH2A mutant cell line was seeded in 200 μl of 10% heatinactivated (HI)FBS/90% DMEM at 200,000 cells per well in a 96-wellplate and nucleofected with 1 μg of sgRNA and 2.5 μg SpCas9 protein asan RNP complex.

sgRNAs used for this assay were purchased as non-modified syntheticsgRNA from Thermo Fisher Scientific. The sgRNAs target the IVS40mutation (FIGS. 5A-5B; 6A, rows 1-5)

The transfected IVS40 USH2A mutant cells were compared to control cells,which are IVS40 USH2A mutant cells transfected with a plasmid thatencodes green fluorescent protein (GFP) (to visually confirmtransfection efficiency), or not transfected at all (data not shown).

Genomic DNA was harvested from the IVS40 USH2A mutant cells 48-96 hoursafter transfection and PCR amplified around the IVS40 mutation of theUSH2A gene. The resulting genome-specific PCR products were thensequenced with a primer located internally in the respective amplifiedPCR product and the sequences were subjected to a TIDE analysis. TIDE isa web tool to rapidly assess genome editing by CRISPR-Cas9 of a targetlocus determined by a guide RNA (gRNA or sgRNA). Based on quantitativesequence trace data from two standard capillary sequencing reactions,the TIDE software quantifies the editing efficacy and identifies thepredominant types of insertions and deletions (indels) in the DNA of atargeted cell pool. See Brinkman et al., Nucl. Acids Res. (2014) for adetailed explanation and examples.

Sequence analysis revealed that the USH2A MP (a sgRNA comprising SEQ IDNO: 5327) that targets the IVS40 mutation had a 69.7% on-target editingefficiency; the USH2A MO (a sgRNA comprising SEQ ID NO: 5321) thattargets the IVS40 mutation had a 68.3% on-target editing efficiency; theUSH2A MG (a sgRNA comprising SEQ ID NO: 5323) that targets the IVS40mutation had a 86.45% on-target editing efficiency; the USH2A MB (asgRNA comprising SEQ ID NO: 5325) that targets the IVS40 mutation had a86.3% on-target editing efficiency; and the USH2A MR (a sgRNA comprisingSEQ ID NO: 5328) that targets the IVS40 mutation had a 91.3% on-targetediting efficiency (FIG. 5B).

These data provide evidence that gRNAs provided herein can directediting within or near the mutant IVS40 USH2A locus. Editing by gRNAssuch as those tested in this Example can contribute to correction of theIVS40 mutation via at least the NHEJ strategy.

Example 10 Testing of sgRNAs (USH2A MO, USH2A MG, USH2A MB, USH2A MP,and USH2A MR)

For selected gRNAs targeting the IVS40 mutation, tests were performed todetermine off-target activity in HEK 293 cells, which are wild-type forUSH2A.

HEK 293 cells were seeded in 200 μl of 10% HIFBS/DMEM at 200,000 cellsper well in a 96-well plate, and nucleofected with 1 μg of sgRNA and 2.5μg SpCas9 protein as an RNP complex.

sgRNAs used for this assay were purchased as non-modified syntheticsgRNA from Thermofisher. The sgRNAs target the IVS40 mutation (FIGS.5A-B)

The transfected HEK 293 cells were compared to control cells, which areHEK 293 cells (Wild-type for USH2A) transfected with a plasmid thatencodes GFP (to visually confirm transfection efficiency), or nottransfected at all (data not shown).

Genomic DNA was harvested from the HEK 293 cells 48-96 hours aftertransfection and PCR amplified around the IVS40 mutation of the USH2Agene. The resulting genome-specific PCR products were then sequencedwith a primer located internally in the respective amplified PCR productand the sequences were subjected to a TIDE analysis.

Sequence analysis revealed that the USH2A MP (a sgRNA comprising SEQ IDNO: 5327) that targets the IVS40 mutation had a 0.7% off-target editingefficiency; the USH2A MO (a sgRNA comprising SEQ ID NO: 5321) thattargets the IVS40 mutation had 1.05% off-target editing efficiency; theUSH2A MG (a sgRNA comprising SEQ ID NO: 5323) that targets the IVS40mutation had a 40.55% off-target editing efficiency; the USH2A MB (asgRNA comprising SEQ ID NO: 5325) that targets the IVS40 mutation had a6.65% off-target editing efficiency; and the USH2A MR (a sgRNAcomprising SEQ ID NO: 5328) that targets the IVS40 mutation had a 1.45%off-target editing efficiency (FIG. 5B).

These data provide evidence that gRNAs provided herein can have minimalwild-type off-target activity and that these gRNAs can be used tocorrect the IVS40 mutation with specificity.

Example 11 Testing of sgRNAs for On-Target Activity in SpCas9-ExpressingHEK 293FT Cells (WT for USH2A)

While the gRNAs characterized in Examples 9 and 10 overlap with theIVS40 mutation when hybridized to their genomic target, another group ofgRNAs that hybridize either upstream or downstream of the IVS40 mutationare also provided herein. gRNAs from this group that were predicted tohave the lowest off-target activity were tested for on-target editingefficiency in wild-type HEK 293FT cells, using SpCas9. Because the gRNAsbind to DNA sequences near, but not overlapping with, the IVS40mutation, Applicants performed these experiments in wild-type HEKFT 293cells.

These HEK 293FT cells with SpCas9 open reading frame (ORF) regulated bya constitutive promoter integrated into the AAVS1 locus were cultured in10% HI FBS/90% DMEM supplemented with 1 μg/ml puromycin and passagedevery 3-4 days.

The HEK 293FT cell line expressing SpCas9 were seeded in 200 μl of 10%HIFBS/DMEM at 50,000 cells per well in a 96-well plate, and transfectedwith 1 μg of sgRNA using Lipofectamine® Messenger-Max™ (available fromThermo Fisher Scientific, Massachusetts, US).

sgRNAs used for this assay were synthesized by in vitro transcription(IVT). The sgRNAs target locations downstream or upstream of the IVS40mutation (FIGS. 6A-B).

At 48 hours post-transfection, culture medium was removed and total DNAwas extracted using prepGem® Tissue Kit (available from VWR,Pennsylvania, US). Part of intron 40 of the USH2A gene (where the gRNAsedit) was PCR amplified. The resulting products were cleaned up usingAMPure XP beads (Available form Beckman Coulter, California, US), andsequenced to assess Cas9-mediated genetic modifications. The frequenciesof small insertions and deletions (indels) were estimated using TIDE.

On-target editing efficiency was determined within intron 40 of theUSH2A gene via TIDE analysis for sgRNAs (a sgRNA comprising any one ofSEQ ID NOs: 5272-5319) (FIGS. 2A-B).

Sequence analysis revealed that the sgRNAs that target locationsdownstream of the IVS40 mutation had an on-target editing efficiencyrange of 24.0 to 89.9% (FIGS. 6A-B).

Sequence analysis revealed that the sgRNAs that target locationsupstream of the IVS40 mutation had an on-target editing efficiency rangeof 24.3 to 91.3% (FIGS. 6A-B).

These data provide evidence that upstream and downstream gRNAs providedherein can display on-target activity at the USH2A locus. This on-targetactivity can contribute to IVS40 mutation correction via at least theexcision strategy and/or the HDR strategy.

Example 12 Testing of sgRNAs for Editing Efficiency in SaCas9 ExpressingK562 Cells (WT for USH2A)

Another group of gRNAs that hybridize either upstream or downstream ofthe IVS40 mutation were tested for on-target activity. These gRNAsassociate with SaCas9 and were used in genetically engineered K562cells. K562 cells express SaCas9 protein from an inducible promoter, andhave the wild-type USH2A gene.

The wild-type K562 cells expressing SaCas9 were treated with 1-10 μg/mLof Doxycycline to induce SaCas9 expression, 48 hours prior totransfection. The cell line was seeded in 200 μl of 10% FBS/IMDM at200,000 cells per well in a 96-well plate, and nucleofected with 1 μgtotal plasmid that encodes sgRNA.

The sgRNAs target locations downstream or upstream of the IVS40 mutation(FIG. 6C).

The edited K562 cells were compared to control cells, which weretransfected with a plasmid that encodes GFP (to visually confirmtransfection efficiency), or not transfected at all (data not shown).

Genomic DNA was harvested from the K562 cells 48-96 hours aftertransfection and PCR amplified around the IVS40 mutation of the USH2Agene. The resulting genome-specific PCR products were then sequencedwith a primer located internally in the respective amplified PCR productand subjected to a TIDE analysis.

Sequence analysis revealed that the sgRNAs that target locationsdownstream of the IVS40 mutation had an on-target editing efficiencyrange of 1.67 to 15.58% (FIG. 6C).

Sequence analysis further revealed that the sgRNAs that target locationsupstream of the IVS40 mutation had an on-target editing efficiency rangeof 1.14 to 30.61% (FIG. 6C).

These data provide evidence that upstream and downstream gRNAs of thepresent disclosure can display on-target activity at the USH2A locus.This on-target activity can contribute to IVS40 mutation correction viaat least the excision strategy and/or the HDR strategy.

Example 13 pET01 Construction

To measure splicing of intron 40 after genome editing according to thepresent disclosure, two plasmids were constructed from pET01. The twoplasmids that pET01 was used to create are: (1) pET01-IVS40 (SEQ ID NO:5525); and (2) pET01-WT (SEQ ID NO: 5524).

pET01 (FIG. 12A) is a plasmid that includes rat pre-proinsulin 5′ and 3′exons separated by an intron sequence. The intron sequence contains amultiple cloning site (MCS). The intron is flanked by a 5′ splice donorsite and a 3′ splice acceptor site of a eukaryotic exon. The 3′spliceacceptor site is followed by a 3′ polyadenylation site (poly A). Thevector contains prokaryotic and eukaryotic genetic elements forreplication in prokaryotic and eukaryotic cells. The expression of thisvector sequence is driven by a promoter present in the long terminalrepeat (LTR) of Rous Sarcoma Virus (RSV) followed by a short stretch ofa eukaryotic gene (phosphatase).

A 3150 bp region of USH2A intron 40 comprising the IVS40 mutation wascloned into the MCS of pET01 to create pET01-IVS40. The IVS40 mutationin the USH2A intron 40 leads to the creation of a splice donor site andinsertion of 152 bp into the USH2A mRNA making the amplified USH2A mRNAfragment 387 total base pairs (FIG. 12B).

A 3150 bp region of USH2A intron 40 comprising the wild-type sequencewas cloned into the MCS of pET01 to create pET01-WT. USH2A mRNA istranscribed from pET01-WT and introns are removed from the mRNA makingthe amplified USH2A mRNA fragment 235 total base pairs (FIG. 12B).

Thus, transcripts from the pET01-IVS40 and the pET01-WT plasmids can beanalyzed to investigate correction of the IVS40 mutation and the statusof splicing in the USH2A gene at intron 40.

Example 14 Splicing Reporter Assay

To measure splicing of intron 40 after genome editing according to thepresent disclosure, splicing of transcripts from the two plasmidsconstructed in Example 13 was investigated using HEK 293 cells thatexpress SpCas9.

Wild-type HEK 293 cells with SpCas9 open reading frame (ORF) regulatedby a constitutive promoter integrated into the AAVS1 locus were culturedin 10% Heat inactivated (HI) FBS/DMEM supplemented with 1 μg/mlpuromycin, and passaged every 3-4 days.

The wild-type HEK 293 cell line expressing SpCas9 was seeded in 200 μlof 10% HIFBS/DMEM at 200,000 cells per well in a 96-well plate, andtransfected with 1 μg of either: (1) a pET01-IVS40; or (2) a pET01-WTand transfected with 1 μg of a sgRNA comprising any one of: USH2A MP(SEQ ID NO: 5327), the USH2A MO (SEQ ID NO: 5321), the USH2A MG (SEQ IDNO: 5323), or the USH2A MB (SEQ ID NO: 5325). sgRNAs used for this assaywere synthesized by in vitro transcription (IVT) by Thermofisher.

RNA was harvested from transfected wild-type HEK 293 cells 48-168 hoursafter transfection and reverse transcribed into cDNA using transcriptspecific primer GATCCACGATGC (SEQ ID NO: 5498), which only amplifiesproducts of plasmid transcription.

The cDNA was then PCR amplified using forward primerGGTGACAGCTGCCAGGATCG (SEQ ID NO: 5499) and reverse primerGCCACCTCCAGTGCCAAGGT (SEQ ID NO: 5500). The PCR products were run on aBioanalyzer (available from Agilent Technologies, California, US), achip-based capillary electrophoresis machine used to analyze RNA.

Bioanalyzer results showed a 235 bp product when wild-type HEK 293 cellswere transfected with pET01-IVS40 and a sgRNA comprising USH2A MP (SEQID NO: 5327) (FIG. 12C, lane 4). These results indicate that the IVS40mutation in the USH2A intron 40 had been edited, which led to adisruption of the splice donor site and removal of 152 bp from the USH2AmRNA fragment making the USH2A mRNA fragment 235 total base pairs.

Bioanalyzer results showed a 235 bp product when wild-type HEK 293 cellswere transfected with pET01-IVS40 and a sgRNA comprising the USH2A MO(SEQ ID NO: 5321) (FIG. 12C, lane 6). These results indicate that theIVS40 mutation in the USH2A intron 40 had been edited, which led to adisruption of the splice donor site and removal of 152 bp from the USH2AmRNA fragment making the USH2A mRNA fragment 235 total base pairs.

Bioanalyzer results showed a 235 bp product when wild-type HEK 293 cellswere transfected with pET01-IVS40 and a sgRNA comprising the USH2A MG(SEQ ID NO: 5323) (FIG. 12C, lane 8). These results indicate that theIVS40 mutation in the USH2A intron 40 had been edited, which led to adisruption of the splice donor site and removal of 152 bp from the USH2AmRNA fragment making the USH2A mRNA fragment less than 235 total basepairs. It is possible that a Bioanalyzer error led to a downward shiftin the band.

Bioanalyzer results showed a 235 bp product when wild-type HEK 293 cellswere transfected with pET01-IVS40 and a sgRNA comprising the USH2A MB(SEQ ID NO: 5325) (FIG. 12C, lane 10). These results indicate that theIVS40 mutation in the USH2A intron 40 had been edited, which led to adisruption of the splice donor site and removal of 152 bp from the USH2AmRNA fragment making the USH2A mRNA fragment 235 total base pairs.

Bioanalyzer results showed a 235 bp product when wild-type HEK 293 cellswere transfected with pET01-WT and a sgRNA comprising USH2A MP (SEQ IDNO: 5327) (FIG. 12C, lane 3). These results indicate that the sgRNAcomprising USH2A MP (SEQ ID NO: 5327) do not affect the RNA splicing ofUSH2A intron 40 comprising a wild-type sequence.

Bioanalyzer results showed a 235 bp product when wild-type HEK 293 cellswere transfected with pET01-WT and a sgRNA comprising the USH2A MO (SEQID NO: 5321) (FIG. 12C, lane 5). These results indicate that the sgRNAcomprising the USH2A MO (SEQ ID NO: 5321) do not affect the RNA splicingof USH2A intron 40 comprising a wild-type sequence.

Bioanalyzer results showed a 235 bp product when wild-type HEK 293 cellswere transfected with pET01-WT and a sgRNA comprising the USH2A MG (SEQID NO: 5323) (FIG. 12C, lane 7). These results indicate that the sgRNAcomprising the USH2A MG (SEQ ID NO: 5323) do not affect the RNA splicingof USH2A intron 40 comprising a wild-type sequence.

Bioanalyzer results showed a 235 bp product when wild-type HEK 293 cellswere transfected with pET01-WT and a sgRNA comprising the USH2A MB (SEQID NO: 5325) (FIG. 12C, lane 9). These results indicate that the sgRNAcomprising the USH2A MB (SEQ ID NO: 5325) do not affect the RNA splicingof USH2A intron 40 comprising a wild-type sequence.

The transfected wild-type HEK 293FT cells expressing SpCas9 werecompared to control cells, which are wild-type HEK 293 cells expressingSpCas9 and transfected with a pET01-IVS40 or pET01-WT, but nottransfected with any sgRNA. Bioanalyzer results showed a 235 bp productwhen wild-type HEK 293 cells were transfected with pET01-WT and no sgRNA(FIG. 12C, lane 1). Bioanalyzer results showed a 387 bp product whenwild-type HEK 293 cells were transfected with pET01-IVS40 and no sgRNA(FIG. 12C, lane 2).

These data provide evidence that gRNAs provided herein can correct thesplicing phenotype associated with the IVS40 mutation at least via theNHEJ strategy. Additionally, the gRNAs do not affect splicing oftranscripts with a USH2A intron 40 comprising a wild-type sequence.

Example 15 Testing of sgRNAs in Cells for Off-Target Activity

It is generally desirable to limit off-target editing. Accordingly, todetermine the extent of off-target editing on a genomic level, gRNAs (orsgRNAs) demonstrated to have on-target activity are tested fortargeted-genome-wide off-target editing using GUIDE-seq, Amplicon-seq,and/or Digenome-seq. Off-target effects are tested in human or non-humanprimate (NHP) photoreceptor cells. Such methods are known in the art andexamples are provided herein.

Example 16 Dual sgRNA Editing Using SpCas9

To further investigate the excision strategy, sgRNAs, that can associatewith SpCas9, were used in pairs to delete the IVS40 mutation withinintron 40 of the USH2A gene (FIG. 7A). “Dual sgRNA editing” refers to afirst sgRNA that targets a location downstream of the IVS40 mutation anda second sgRNA that targets a location upstream of the IVS40 mutation.

FIGS. 7B-F show 5 possible editing outcomes from using dual sgRNAs wherethe first sgRNA binds/targets a location upstream of the IVS40 mutationand the second sgRNA binds/targets a location downstream of the IVS40mutation. The 5 possible editing outcomes include: (1) the genomic DNAremains unedited (FIG. 7B); (2) the genomic DNA is edited at a locationupstream of the IVS40 mutation or at a location downstream of the IVS40mutation (FIG. 7C); (3) the genomic DNA is edited at both a locationupstream of the IVS40 mutation and at a location downstream of the IVS40mutation, but editing does not result in a deletion (beyond any expectedindels) (FIG. 7D); (4) the genomic DNA is edited at both a locationupstream of the IVS40 mutation and at a location downstream of the IVS40mutation, and editing results in a deletion (FIG. 7E); and (5) thegenomic DNA is edited at both a location upstream of the IVS40 mutationand at a location downstream of the IVS40 mutation, but editing does notresult in a deletion. Instead, editing results in an inversion (FIG.7F).

The size of the deletion product (in the case of FIG. 7E) and editingefficiency is important for each of the dual sgRNAs. The size of thedeletion products generated from edits between the first sgRNA andsecond sgRNA was calculated from theoretically predicted DNA break siteslocated three nucleotides upstream of the PAM sequence for each of thefirst sgRNAs and second sgRNAs. For the dual sgRNAs that associate withSpCas9, the size of the deletion products ranged from 70 to 1422 bp(Table 4, FIG. 9A).

TABLE 4 Guide RNA Name Guide RNA Name Deletion size (Upstream of IVS40)(Downstream of IVS40) (bp) USH2Amut_T387 USH2Amut_T715 70 USH2Amut_T387USH2Amut_T261 117 USH2Amut_T387 USH2Amut_T343 137 USH2Amut_T176USH2Amut_T715 198 USH2Amut_T210 USH2Amut_T715 199 USH2Amut_T176USH2Amut_T261 245 USH2Amut_T210 USH2Amut_T261 246 USH2Amut_T176USH2Amut_T343 265 USH2Amut_T210 USH2Amut_T343 266 USH2Amut_T193USH2Amut_T715 304 USH2Amut_T505 USH2Amut_T715 356 USH2Amut_T193USH2Amut_T261 361 USH2Amut_T193 USH2Amut_T343 381 USH2Amut_T505USH2Amut_T261 403 USH2Amut_T505 USH2Amut_T343 423 USH2Amut_T585USH2Amut_T715 518 USH2Amut_T585 USH2Amut_T261 565 USH2Amut_T585USH2Amut_T343 585 USH2Amut_T6 USH2Amut_T715 676 USH2Amut_T6USH2Amut_T261 723 USH2Amut_T6 USH2Amut_T343 743 USH2Amut_T193USH2Amut_T9 1050 USH2Amut_T505 USH2Amut_T9 1102 USH2Amut_T585USH2Amut_T9 1264 USH2Amut_T6 USH2Amut_T9 1422

Wild-type HEK 293 cells with SpCas9 open reading frame (ORF) regulatedby a constitutive promoter integrated into the AAVS1 locus were culturedin 10% Heat inactivated (HI) FBS/DMEM supplemented with 1 μg/mlpuromycin and passaged every 3-4 days. The use of wild-type HEK 293cells (which express SpCas9) for this experiment is possible because thedual sgRNAs bind to regions upstream and downstream of the IVS40mutation and these regions do not differ between the wild-type cell lineand the IVS40 USH2A mutant cell line. Genetically engineered HEK 293cells that have a homozygous C.7595-2144A>G mutation (IVS40 USH2A mutantcell line) could be used for these experiments, but these cells do notexpress SpCas9.

The wild-type HEK 293 cell line expressing SpCas9 was seeded in 200 μlof 10% HIFBS/DMEM at 200,000 cells per well in a 96-well plate andtransfected with 0.5 μg of a first sgRNA that targets a locationupstream of the IVS40 mutation and 0.5 μg of a second sgRNA that targetsa location downstream of the IVS40 mutation.

sgRNAs targeting a location upstream or downstream of the IVS40 mutationthat were used for this assay were synthesized by Thermofisher. ThesesgRNAs comprise a specific protospacer sequence and ThermoFisher'sproprietary tracrRNA sequence.

The transfected wild-type HEK 293 cells expressing SpCas9 were comparedto control cells, which are wild-type HEK 293 cells expressing SpCas9,but not transfected with any sgRNA, or were transfected with aGFP-encoding plasmid (data not shown).

TIDE analysis cannot be used to measure editing efficiency when dualsgRNAs are used since a large region of genomic DNA is excised and theTIDE software cannot quantify these larger deletions. TIDE analysis isbetter suited to analyze small sized deletions and insertions, such asthose expected to be generated via NHEJ resulting from a DSB generatedby a single sgRNA. In addition, shorter length PCR products would befavored in a PCR reaction to estimate the editing level of dual sgRNAs,thus overestimating the percentage of edited cells. Instead,quantitative analysis of deletions generated from edits between thefirst sgRNA and second sgRNA can be best performed using droplet digital(dd)PCR.

Genomic DNA was harvested from transfected wild-type HEK 293 cells 48-96hours after transfection, digested with BamHI restriction enzyme, and 70ng was used for ddPCR. During ddPCR, two PCR products (a first PCRproduct and second PCR product) could be amplified (FIG. 8). To reducethe viscosity before ddPCR, the genomic DNA was digested with BamHI,which does not cut in the immediate vicinity of the two PCR products orthe regions between them.

The first PCR product (the target PCR product) was amplified using aforward primer CCAGAGCAGGAAGCTAATAAA (SEQ ID NO: 5501) and a reverseprimer GATGAACTTGCACTTCAAACC (SEQ ID NO: 5503). The target PCR productwas bound by a USH2A target probe labeled with 6-Carboxyfluorescein(FAM) AATTGAACACCTCTCCTTTCCCAAG (SEQ ID NO: 5502). In unedited cells, nogenomic DNA was deleted, the forward and reverse primers amplified thegenomic DNA creating a target PCR product, and the USH2A target probe(TaqMan type) labeled/quantified the target PCR product. In editedcells, part of the genomic DNA was deleted and the USH2A target probecannot bind the edited DNA because the probe's binding site has beenexcised.

A second PCR product (the reference PCR product) was amplified usingforward primer ACCTACCTATGTTACCACTCA (SEQ ID NO: 5504) and reverseprimer GTCACCTTCTCTTACCTCAAAT (SEQ ID NO: 5506). The reference PCRproduct was bound by a USH2A reference probe labeled with2′-chloro-7′phenyl-1,4-dichloro-6-carboxy-fluorescein (VIC)CTTAGTGGAATCACAGACAATGGGC (SEQ ID NO: 5505). In both edited and uneditedcells, that genomic area was unaffected, the forward and reverse primersamplify the genomic DNA creating a reference PCR product, and the USH2Areference probe labeled/quantified the reference PCR product. For thisreason, the amount of reference PCR product should not vary betweenedited and unedited cells.

In unedited cell, a comparison of the ratio of target PCR product toreference PCR product should be stable, and ideally is close to 1(assuming that there is similar efficiency of amplification for the twoPCR products). In edited cells, the probe does not bind to the targetPCR product DNA because that target site within the genome has beenexcised; therefore the ratio of target PCR product to reference PCRproduct decreases, e.g., compared to the ratio in unedited cells.

The editing efficiency of selected dual sgRNAs that associate withSpCas9 was determined to be in the range of 18.5 to 66.3% (FIGS. 9A-B).

These data provide evidence that the excision strategy provided herein,using dual sgRNAs, can cause a deletion of the DNA encoding the IVS40mutation. The data suggest that splicing of the USH2A gene transcriptcan be corrected.

Example 17 Dual sgRNA Editing Using SaCas9

To further investigate the excision strategy, sgRNAs, that can associatewith SaCas9, were used in pairs to delete the IVS40 mutation withinintron 40 of the USH2A gene (FIG. 7A). “Dual sgRNA editing” refers to afirst gRNA that targets a location downstream of the IVS40 mutation anda second sgRNA that targets a location upstream of the IVS40 mutation.

The size of the deletion product (in the case of FIG. 7E) and theediting efficiency is important for each of the dual sgRNAs. The size ofthe deletion products generated from edits between the first sgRNA andsecond sgRNA was calculated from theoretically predicted DNA break siteslocated three nucleotides upstream of the PAM sequence for each of thefirst sgRNAs and second sgRNAs. For the dual sgRNAs that associate withSaCas9, the size of the deletion products ranged from 211 to 1078 bp(Table 5, FIG. 11A).

TABLE 5 Guide RNA Name Guide RNA Name Deletion size (Upstream of IVS40)(Downstream of IVS40) (bp) USH2Amut_T93 USH2Amut_T127 211 USH2Amut_T93USH2Amut_T115 275 USH2Amut_T93 USH2Amut_T215 400 USH2Amut_T140USH2Amut_T127 424 USH2Amut_T142 USH2Amut_T127 429 USH2Amut_T134USH2Amut_T127 459 USH2Amut_T93 USH2Amut_T131 481 USH2Amut_T140USH2Amut_T115 488 USH2Amut_T142 USH2Amut_T115 493 USH2Amut_T134USH2Amut_T115 523 USH2Amut_T190 USH2Amut_T127 560 USH2Amut_T140USH2Amut_T215 613 USH2Amut_T142 USH2Amut_T215 618 USH2Amut_T190USH2Amut_T115 624 USH2Amut_T134 TUSH2Amut_215 648 USH2Amut_T140USH2Amut_T131 694 USH2Amut_T142 USH2Amut_T131 699 USH2Amut_T134USH2Amut_T131 729 USH2Amut_T93 USH2Amut_T3 729 USH2Amut_T190USH2Amut_T215 749 USH2Amut_T190 USH2Amut_T131 830 USH2Amut_T140USH2Amut_T3 942 USH2Amut_T142 USH2Amut_T3 947 USH2Amut_T134 USH2Amut_T3977 USH2Amut_T190 USH2Amut_T3 1078

Wild-type K562 cells contain two wild-type alleles of the USH2A gene,and the cells were engineered to stably express Staphylococcus aureusCas9 endonuclease under a Doxycycline inducible promoter. The use ofwild-type K562 cells (which express SaCas9) for this experiment ispossible because the dual sgRNAs bind to regions upstream and downstreamof the IVS40 mutation, and these regions do not differ between thewild-type cell line and the IVS40 USH2A mutant cell line. Geneticallyengineered HEK 293 cells that have a homozygous C.7595-2144A>G mutation(IVS40 USH2A mutant cell line) could be used, but these cells do notexpress SaCas9.

The wild-type K562 cells expressing SaCas9 were treated with 1-10 μg/mLof Doxycycline to induce SaCas9 expression, 48 hours prior totransfection. The wild-type K562 cells expressing SaCas9 were thenseeded in 200 μl of 10% FBS/IMDM at 200,000 cells per well in a 96-wellplate, and nucleofected with 1 μg of a plasmid that encodes a firstsgRNA that targets a location upstream of the IVS40 mutation (FIG. 10;or any one of SEQ ID NOs: 5507-5522 and SEQ ID NOs: 5551-5557) and 1 μgof a plasmid that encodes a second sgRNA that targets a locationdownstream of the IVS40 mutation (FIG. 10; or any one of SEQ ID NOs:5507-5522 and SEQ ID NOs: 5551-5557).

The transfected wild-type K562 cells expressing SaCas9 were compared tocontrol cells, which are wild-type K562 cells expressing SaCas9, but nottransfected with any plasmids that encode sgRNA (data not shown).

The transfected wild-type K562 cells were treated with 1-10 μg/mLDoxycycline for an additional 48-96 hours post-transfection. Genomic DNAwas harvested from transfected wild-type K562 cells 48-96 hourspost-transfection, digested with BamHI restriction enzyme, and 40-70 ngwas used for ddPCR analysis as described in Example 16.

The editing efficiency of several dual sgRNAs that associate with SaCas9was determined to be in the range of 8.7 to 30.8% (FIGS. 11A-B).

These data provide evidence that the excision strategy provided herein,using dual sgRNAs, can cause a deletion of the DNA encoding the IVS40mutation. The data suggest that splicing of the USH2A gene transcriptwas corrected.

Example 18 Construction of a Cell Line for Measuring Splicing Via BFPExpression

To enable direct comparisons between multiple combinations of gRNAs andCas9 orthologs, Applicants designed a blue fluorescent protein (BFP)splicing reporter assay based on expression of BFP. HEK 293 cells wereused to create the cell line needed for the BFP splicing reporter assay.

A Jump-In™ GripTite™ HEK 293 Kit (available from Thermo FisherScientific) was used to integrate a reporter construct into the HEK 293genome. This kit comprises HEK 293 cells comprising an R4 attP site thatcan be targeted by an R4 integrase to promote integration of thereporter construct into the cellular genome at an unspecified site. Aplasmid encoding the integrase and a second plasmid engineered to carrythe reporter construct were co-transfected into cells. Afterintegration, cells comprising the reporter construct were selected using10 μg/mL blasticidin.

The integrated reporter construct comprises a phosphoglycerate kinasepromoter operably linked to a BFP gene to allow for transcription of theBFP gene. The BFP gene comprises part of intron 40 of the USH2A gene.Two versions of the construct exist. A first version (the “wild-typeversion”) comprises a wild-type intron 40 sequence (FIG. 13A). Thewild-type version served to validate the BFP splicing reporter assay andconfirm that the construct could express BFP (data not shown). A secondversion (the “IVS40 mutant version”) comprises an IVS40 mutant intron 40sequence (FIGS. 13B-C).

FIGS. 13B-C show a diagram of the how results from the BFP splicingreporter assay can be interpreted. A cell comprising the IVS40 mutantversion of the construct will not express detectable levels of BFP.Although the phosphoglycerate kinase promoter activates transcription ofthe BFP gene, the IVS40 mutation in the intron 40 sequence causesaberrant splicing and the inclusion of the flanking, intronic sequenceof IVS40, which makes the reporter BFP gene out of frame (FIG. 13B).However, if genome editing according to the present disclosure correctsthe aberrant mRNA splicing via deletion of the IVS40 mutation or viamodification of a sequence within or near the IVS40 mutation, then BFPcan be translated in-frame to produce a functional protein (FIG. 13C).BFP expression can then be detected by flow cytometry or another methodsuitable to detect protein expression. The BFP splicing reporter assaycan indirectly estimate the effects (and rate thereof) of genome editingmediated by Cas9 orthologs and their corresponding gRNAs on USH2A IVS40mutant splicing.

Example 19 BFP Splicing Reporter Assay Using SpCas9 Guide RNAs

To test the ability of gRNAs paired with SpCas9 to correct aberrantsplicing due to the IVS40 mutation, a BFP splicing reporter assay wasperformed. Both the NHEJ strategy and the excision strategy were tested.

Engineered HEK 293 cells from Example 18 comprising the IVS40 mutantversion of the construct described in Example 18 were used during theBFP splicing reporter assay. Engineered HEK 293 Cells were grown in 90%DMEM/10% dialyzed FBS supplemented with GlutaMAX™ (available from ThermoFisher Scientific), 25 mM HEPES buffer (pH=7.3), and 0.1 mM MEMnon-essential amino acids solution.

In some assays, single sgRNAs that overlap with the IVS40 mutation wereused. These assays tested the NHEJ strategy. In other assays, dualsgRNAs were used. These assays tested the excision strategy.

Regardless of whether a single sgRNA or dual sgRNAs were used, eachsgRNA was encoded on a separate plasmid comprising (1) the sgRNA gene,operably linked to a U6 promoter and (2) a SpCas9 gene, operably linkedto a chicken beta-actin promoter. Thus, in single sgRNA experiments,cells were transfected with one plasmid, and, in dual sgRNA experiments,cells were transfected with two plasmids. Transfections usedLipofectamine® 3000 (available from Thermo Fisher Scientific). Tocontrol for gene dosage, 1 μg of each plasmid was transfected when twoplasmids were transfected and 2 μg of plasmid were transfected when asingle plasmid was transfected. Each plasmid also encodes a GFP geneoperably linked to Cas9 genes through T2A peptide. This can allow formonitoring of the transfection efficiency via measurement of GFP signal.

150,000 cells were transfected in each assay and incubated for 72 hoursprior to fluorescence level analysis by flow cytometry. As a negativecontrol for editing, transfection of cells with a plasmid that encodesSpCas9 and a scrambled sgRNA that does not edit intron 40 of the humanUSH2A gene was also performed (data not shown). Results are shown inTable 6 and FIG. 14. Values reported are the percentage of total livecells that were BFP positive. All assays were performed in triplicate.

TABLE 6 overlapping Deletion sgRNA or sgRNA sgRNA upstream Average BFPSize downstream of the of the IVS40 BFP Standard (bp) IVS40 mutationmutation signal deviation N/A USH2A MO N/A 22.86833 2.6 N/A USH2A MP N/A24.41667 2.7 N/A USH2A MR N/A 13.83 2.1  70 USH2Amut_T715 USH2Amut_T38716.20333 2.1 117 USH2Amut_T261 USH2Amut_T387 11.17167 5.5 137USH2Amut_T343 USH2Amut_T387 12.66667 1.9 198 USH2Amut_T715 USH2Amut_T17613.12167 3.7 199 USH2Amut_T715 USH2Amut_T210 17.76167 4.1 266USH2Amut_T343 USH2Amut_T210 8.846667 1.9 304 USH2Amut_T715 USH2Amut_T19314.36 1.6 356 USH2Amut_T715 USH2Amut_T505 17.06167 3.6 381 USH2Amut_T343USH2Amut_T193 9.503333 4.4 518 USH2Amut_T715 USH2Amut_T585 13.98 3.2

When engineered HEK 293 cells were transfected with a plasmid comprisinga sgRNA gene encoding USH2A MO (a sgRNA comprising SEQ ID NO: 5321),splicing of the BFP reporter was corrected by genome editing leading toBFP expression in 22.9% of cells.

When engineered HEK 293 cells were transfected with a plasmid comprisinga sgRNA gene encoding USH2A MP (a sgRNA comprising SEQ ID NO: 5327),splicing of the BFP reporter was corrected by genome editing leading toBFP expression in 24.4% of cells.

When engineered HEK 293 cells were transfected with a plasmid comprisinga sgRNA gene encoding USH2A MR (a sgRNA comprising SEQ ID NO: 5328),splicing of the BFP reporter was corrected by genome editing leading toBFP expression in 13.8% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T715 (a sgRNA comprisingSEQ ID NO: 5300) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T387 (a sgRNA comprising SEQ ID NO: 5295), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 16.2% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T261 (a sgRNA comprisingSEQ ID NO: 5298) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T387 (a sgRNA comprising SEQ ID NO: 5295), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 11.2% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T343 (a sgRNA comprisingSEQ ID NO: 5279) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T387 (a sgRNA comprising SEQ ID NO: 5295), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 12.7% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T715 (a sgRNA comprisingSEQ ID NO: 5300) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T176 (a sgRNA comprising SEQ ID NO: 5290), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 13.1% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T715 (a sgRNA comprisingSEQ ID NO: 5300) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T210 (a sgRNA comprising SEQ ID NO: 5294), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 17.8% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T343 (a sgRNA comprisingSEQ ID NO: 5279) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T210 (a sgRNA comprising SEQ ID NO: 5294), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 8.8% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T715 (a sgRNA comprisingSEQ ID NO: 5300) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T193 (a sgRNA comprising SEQ ID NO: 5277), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 14.4% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T715 (a sgRNA comprisingSEQ ID NO: 5300) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T505 (a sgRNA comprising SEQ ID NO: 5274), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 17.1% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T343 (a sgRNA comprisingSEQ ID NO: 5279) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T193 (a sgRNA comprising SEQ ID NO: 5277), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 9.5% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T715 (a sgRNA comprisingSEQ ID NO: 5300) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T585 (a sgRNA comprising SEQ ID NO: 5275), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 14.0% of cells.

These data provide evidence that splicing of transcripts from a USH2Agene comprising an IVS40 mutation can be corrected via either the NHEJstrategy provided herein or the excision strategy provided herein andsuggest that functional USH2A protein can be expressed.

Example 20 BFP Splicing Reporter Assay Using SaCas9 Guide RNAs

To test the ability of gRNAs paired with SaCas9 to correct aberrantsplicing due to the IVS40 mutation, a BFP splicing reporter assay wasperformed. The excision strategy was tested.

Engineered HEK 293 cells from Example 18 comprising the IVS40 mutantversion of the construct described in Example 18 were used during theBFP splicing reporter assay. Engineered HEK 293 Cells were grown as inExample 19. Dual sgRNAs were used to test the excision strategy.

Each sgRNA was encoded on a separate plasmid comprising the (1) sgRNAgene, operably linked to a U6 promoter and (2) a SaCas9 gene, operablylinked to a chicken beta-actin promoter. Thus, cells were transfectedwith two plasmids (one plasmid for each of the pair of dual sgRNAs to betested). Transfections used Lipofectamine® 3000 (available from ThermoFisher Scientific). To control for gene dosage, cells were transfectedwith 1 μg of each plasmid. Each plasmid also encodes a GFP gene operablylinked to a Cas9 through T2A peptide. This can allow for monitoring ofthe transfection efficiency via measurement of GFP signal.

150,000 cells were transfected in each assay and incubated for 72 hoursprior to fluorescence level analysis by flow cytometry. As a negativecontrol for editing, transfection of cells with a plasmid that encodesSaCas9 and a scrambled sgRNA that does not edit intron 40 of the humanUSH2A gene was also performed (data not shown). Results are shown inTable 7 and FIG. 15. Values reported are the percentage of total livecells that were BFP positive. All assays were performed in triplicate.

TABLE 7 sgRNA Deletion downstream of sgRNA upstream Average BFP Size theIVS40 of the IVS40 BFP Standard (bp) mutation mutation signal deviation275 USH2Amut_T93 USH2Amut_T115 27.8 10.1 459 USH2Amut_T134 USH2Amut_T12722.2 11.8 488 USH2Amut_T140 USH2Amut_T115 35.5 10.8 493 USH2Amut_T142USH2Amut_T115 33.5 10.9 523 USH2Amut_T134 USH2Amut_T115 25.6 11.7 624USH2Amut_T190 USH2Amut_T115 35.1 12.3 648 USH2Amut_T134 USH2Amut_T21526.6 9.5 694 USH2Amut_T140 USH2Amut_T131 33.2 10.2 729 USH2Amut_T134USH2Amut_T131 27.4 11.2 749 USH2Amut_T190 USH2Amut_T215 24.3 10.2 830USH2Amut_T190 USH2Amut_T131 27.7 9.1

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T93 (a sgRNA comprisingSEQ ID NO: 5448) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T115 (a sgRNA comprising SEQ ID NO: 5449), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 27.8% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T134 (a sgRNA comprisingSEQ ID NO: 5452) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T127 (a sgRNA comprising SEQ ID NO: 5450), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 22.2% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T140 (a sgRNA comprisingSEQ ID NO: 5453) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T115 (a sgRNA comprising SEQ ID NO: 5449), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 35.5% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T142 (a sgRNA comprisingSEQ ID NO: 5454) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T115 (a sgRNA comprising SEQ ID NO: 5449), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 33.5% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T142 (a sgRNA comprisingSEQ ID NO: 5454) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T115 (a sgRNA comprising SEQ ID NO: 5449), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 33.5% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T134 (a sgRNA comprisingSEQ ID NO: 5452) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T115 (a sgRNA comprising SEQ ID NO: 5449), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 25.6% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T190 (a sgRNA comprisingSEQ ID NO: 5455) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T115 (a sgRNA comprising SEQ ID NO: 5449), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 35.1% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T134 (a sgRNA comprisingSEQ ID NO: 5452) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T215 (a sgRNA comprising SEQ ID NO: 5457), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 26.6% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T140 (a sgRNA comprisingSEQ ID NO: 5453) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T131 (a sgRNA comprising SEQ ID NO: 5451), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 33.2% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T134 (a sgRNA comprisingSEQ ID NO: 5452) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T131 (a sgRNA comprising SEQ ID NO: 5451), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 27.4% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T190 (a sgRNA comprisingSEQ ID NO: 5455) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T215 (a sgRNA comprising SEQ ID NO: 5457), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 24.3% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T190 (a sgRNA comprisingSEQ ID NO: 5455) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T131 (a sgRNA comprising SEQ ID NO: 5451), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 27.7% of cells.

These data provide evidence that splicing of transcripts from a USH2Agene comprising an IVS40 mutation can be corrected via the excisionstrategy provided herein and suggest that functional USH2A protein canbe expressed.

Example 21 BFP Splicing Reporter Assay for Selected Combinations ofSaCas9, SpCas9, and gRNAs

To test the ability of gRNAs paired with SaCas9 or SpCas9 to correctaberrant splicing due to the IVS40 mutation, a BFP splicing reporterassay was performed. These experiments allowed comparisons to be madeamong various gRNA and nuclease combinations using data from a singleexperiment. Both the NHEJ strategy and the excision strategy weretested.

Engineered HEK 293 cells from Example 18 comprising the IVS40 mutantversion of the construct described in Example 18 were used during theBFP splicing reporter assay. Engineered HEK 293 Cells were grown as inExample 19.

In some assays, single sgRNAs that overlap with the IVS40 mutation wereused.

These assays tested the NHEJ strategy. In other assays, dual sgRNAs wereused. These assays tested the excision strategy.

Regardless of whether a single sgRNA or dual sgRNAs were used, eachsgRNA was encoded on a separate plasmid comprising (1) the sgRNA gene,operably linked to a U6 promoter and (2) a SpCas9 or SaCas9 gene,operably linked to a chicken beta-actin promoter. Thus, in single sgRNAexperiments, cells were transfected with one plasmid, and, in dual sgRNAexperiments, cells were transfected with two plasmids. Transfectionsused Lipofectamine® 3000 (available from Thermo Fisher Scientific). Tocontrol for gene dosage, 1 μg of each plasmid was transfected when twoplasmids were transfected and 2 μg of plasmid were transfected when asingle plasmid was transfected. Each plasmid also encodes a GFP geneoperably linked to a separate promoter. This can allow for monitoring ofthe transfection efficiency via measurement of GFP signal. GFP positivecells are cells which were successfully transfected with at least oneplasmid.

100,000-150,000 cells were seeded in 1 mL of medium 24 hours beforetransfection. After transfection, cells were incubated for 72 hoursprior to fluorescence level analysis by flow cytometry. As a negativecontrol for editing, transfection of cells with a plasmid that encodesSaCas9 and a non-targeting sgRNA that does not edit intron 40 of thehuman USH2A gene was also performed. A similar negative control wasperformed with a plasmid that encodes SpCas9 and a non-targeting sgRNA.Results are shown in Table 8 and FIG. 16. Values reported are thepercentage of total GFP positive cells that were BFP positive.

TABLE 8 sgRNA downstream of Deletion the IVS40 mutation or sgRNAupstream of Nuclease Size (bp) overlapping sgRNA the IVS40 mutationAverage SaCas9 N/A SaCas9 vector N/A 0.00 backbone negative controlSaCas9 275 USH2Amut_T93 USH2Amut_T115 36.02 SaCas9 488 USH2Amut_T140USH2Amut_T115 50.73 SaCas9 493 USH2Amut_T142 USH2Amut_T115 49.14 SaCas9523 USH2Amut_T134 USH2Amut_T115 36.11 SaCas9 624 USH2Amut_T190USH2Amut_T115 48.33 SaCas9 694 USH2Amut_T140 USH2Amut_T131 45.76 SpCas9N/A SpCas9 vector 0.00 Backbone Control SpCas9 N/A UHS2A MO 37.97 SpCas9N/A USH2A MP 34.95 SpCas9  70 USH2Amut_T715 USH2Amut_T387 17.02 SpCas9137 USH2Amut_T343 USH2Amut_T387 11.66 SpCas9 198 USH2Amut_T715USH2Amut_T176 20.47 SpCas9 304 USH2Amut_T715 USH2Amut_T193 29.22 SpCas9356 USH2Amut_T715 USH2Amut_T505 27.86

The following data were obtained for sgRNAs expressed along with SaCas9:

When engineered HEK 293 cells were transfected with a first plasmidcomprising the vector backbone negative control, splicing of the BFPreporter was corrected by genome editing leading to BFP expression in0.0% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T93 (a sgRNA comprisingSEQ ID NO: 5448) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T115 (a sgRNA comprising SEQ ID NO: 5449), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 36.0% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T140 (a sgRNA comprisingSEQ ID NO: 5453) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T115 (a sgRNA comprising SEQ ID NO: 5449), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 50.7% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T142 (a sgRNA comprisingSEQ ID NO: 5454) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T115 (a sgRNA comprising SEQ ID NO: 5449), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 49.1% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T134 (a sgRNA comprisingSEQ ID NO: 5452) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T115 (a sgRNA comprising SEQ ID NO: 5449), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 36.1% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T190 (a sgRNA comprisingSEQ ID NO: 5455) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T115 (a sgRNA comprising SEQ ID NO: 5449), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 48.3% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T140 (a sgRNA comprisingSEQ ID NO: 5453) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T131 (a sgRNA comprising SEQ ID NO: 5451), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 45.8% of cells.

The following data were obtained for sgRNAs expressed along with SpCas9:

When engineered HEK 293 cells were transfected with a first plasmidcomprising the vector backbone negative control, splicing of the BFPreporter was corrected by genome editing leading to BFP expression in0.0% of cells.

When engineered HEK 293 cells were transfected with a plasmid comprisinga sgRNA gene encoding USH2A MO (a sgRNA comprising SEQ ID NO: 5321),splicing of the BFP reporter was corrected by genome editing leading toBFP expression in 38.0% of cells.

When engineered HEK 293 cells were transfected with a plasmid comprisinga sgRNA gene encoding USH2A MP (a sgRNA comprising SEQ ID NO: 5327),splicing of the BFP reporter was corrected by genome editing leading toBFP expression in 35.0% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T715 (a sgRNA comprisingSEQ ID NO: 5300) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T387 (a sgRNA comprising SEQ ID NO: 5295), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 17.0% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T343 (a sgRNA comprisingSEQ ID NO: 5279) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T387 (a sgRNA comprising SEQ ID NO: 5295), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 11.7% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T715 (a sgRNA comprisingSEQ ID NO: 5300) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T176 (a sgRNA comprising SEQ ID NO: 5290), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 20.5% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T715 (a sgRNA comprisingSEQ ID NO: 5300) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T193 (a sgRNA comprising SEQ ID NO: 5277), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 29.2% of cells.

When engineered HEK 293 cells were transfected with a first plasmidcomprising a first sgRNA gene encoding USH2Amut_T715 (a sgRNA comprisingSEQ ID NO: 5300) and a second plasmid comprising a second sgRNA geneencoding USH2Amut_T505 (a sgRNA comprising SEQ ID NO: 5274), splicing ofthe BFP reporter was corrected by genome editing leading to BFPexpression in 27.9% of cells.

These data provide evidence that splicing of transcripts from a USH2Agene comprising an IVS40 mutation can be corrected via either the NHEJstrategy provided herein or the excision strategy provided herein,leading to expression of functional USH2A protein.

Example 22 ddPCR Assay for Selected SpCas9 gRNAs

To test the ability of gRNAs paired with SpCas9 to correct the IVS40mutation, a droplet digital reverse transcriptase PCR (ddPCR) experimentwas performed. Both the NHEJ strategy and the excision strategy weretested.

The IVS40 USH2A mutant cell line generated in Example 8 was used inthese experiments. The IVS40 USH2A mutant cell line was seeded in 200 μlof 10% heat inactivated (HI) FBS/90% DMEM at 200,000 cells per well in a96-well plate.

In some experiments, a single, unmodified, synthetic sgRNA (obtainedfrom Thermo Fisher Scientific) was used. These experiments tested theNHEJ strategy. In other experiments, dual, unmodified, synthetic sgRNA(obtained from Thermo Fisher Scientific) were used (dual sgRNAs). Theseexperiments tested the excision strategy.

When a single sgRNA was used, nucleofection was performed with 1 μg ofsgRNA and 2.5 μg of SpCas9 protein as an editing RNP complex. When dualsgRNAs were used, nucleofection was performed with 0.5 μg of each sgRNAand 2.5 μg of SpCas9 protein as editing RNP complexes.

Applicants, desired to perform ddPCR on cells transfected with editingRNA complex(es). However, the USH2A gene is transcribed in the IVS40USH2A mutant cell genetic background at low levels such thattranscriptional activation of the USH2A gene was preferred before mRNAwas isolated. To increase transcription of USH2A mRNA, the cells weretransfected a second time (72 hours after the first transfection) withplasmids comprising genes encoding a USH2A transcriptional activationcomplex. A first plasmid encodes a dCas9 variant (a Cas9 proteincomprising point mutations which inactivate nuclease activity). A secondplasmid encodes a sgRNA and bacteriophage MS2 coat protein fused to theC-terminal portion of the p65 subunit of mouse NF-κB (p65). The sgRNAguides the RNA complex to a region upstream of the USH2A transcriptionalstart site.

The dCas9 is fused to the transcriptional activator VP64. The sgRNA isfused to two MS2 RNA aptamers, which can recruit transactivators fusedto MS2 coat protein (e.g., p65, and HSF1). When the RNP complex isassembled upstream of the USH2A transcriptional start site, the activityof the fusion partners and cellular proteins can activate transcriptionof the USH2A gene, thereby increasing the amount mRNA that is producedin the previously edited IVS40 USH2A mutant cells.

7 days after the second transfection, mRNA was isolated from the cellsand cDNA was prepared by reverse transcription. ddPCR was performed onthe cDNA to quantify transcripts made from USH2A genes where the IVS40mutation was deleted or modified versus transcripts made from USH2Agenes where the IVS40 mutation was not deleted or modified.

During the ddPCR, A first PCR product can be amplified using a forwardprimer CCAACCGTACACAGAGTATATG (SEQ ID NO: 5558) and a reverse primerCTTGACTTCACATCCAGAAGAA (SEQ ID NO: 5559). The first PCR product is shownin FIG. 17A and is produced from cDNA resulting from mRNA transcribedfrom USH2A genes where the IVS40 mutation was not deleted or modified.The first PCR product can be bound by a first probe (“Mutant SpecificProbe” in FIG. 17A). The first probe is labeled with2′-chloro-7′phenyl-1,4-dichloro-6-carboxy-fluorescein (VIC) andcomprises the sequence AGTAGATTCGCTGCTCTTGTTGC (SEQ ID NO: 5560).

During the ddPCR, a second PCR product can be amplified using theforward primer CCAACCGTACACAGAGTATATG (SEQ ID NO: 5558) and the reverseprimer CTTGACTTCACATCCAGAAGAA (SEQ ID NO: 5559). The second PCR productis shown in FIG. 17B and is produced from cDNA resulting from mRNAtranscribed from USH2A genes where the IVS40 mutation was deleted ormodified. The second PCR product can be bound by a second probe (“WildType Specific Probe” in FIG. 17B). The second probe is labeled with6-Carboxyfluorescein (FAM) and comprises the sequenceAGAGGACAAACCTGGACCTGTAG (SEQ ID NO: 5561). The percentage of the totalsignal detected during the ddPCR which is from the first, VIC-labeledprobe corresponds to the fraction of mRNA produced from USH2A geneswhere the IVS40 mutation was not deleted or modified (% uncorrectedUSH2A transcript,

FIG. 18). The percentage of the total signal detected during the ddPCRwhich is from the second, FAM-labeled probe corresponds to the fractionof mRNA produced from USH2A genes where the IVS40 mutation was deletedor modified (% corrected USH2A transcript, FIG. 18).

Results are shown in Table 9 and FIG. 18.

TABLE 9 % % corrected uncorrected Deletion USH2A USH2A Dual SpCas9sgRNAs Size (bp) transcript transcript USH2Amut_T176 and 198 77.5 22.5USH2Amut_T715 USH2Amut_T210 and 199 67.3 32.7 USH2Amut_T715USH2Amut_T387 and  70 77.6 22.4 USH2Amut_T715 USH2Amut_T193 and 381 62.637.4 USH2Amut_T343 USH2Amut_T387 and 137 77.4 22.6 USH2Amut_T343USH2Amut_T505 and 423 48.5 51.5 USH2Amut_T343 USH2Amut_T585 and 585 71.928.1 USH2Amut_T343 No RNP (negative control) N/A 1.0 99.0 % % correcteduncorrected USH2A USH2A Single SpCas9 Guide transcript transcript USH2AMP N/A 90.7 9.3 USH2A MO N/A 89.8 10.2 USH2A MG N/A 37.4 62.6 USH2A MBN/A 43.3 56.7 USH2A MR N/A 84.1 15.9 GFP plasmid control N/A 1.9 98.1mCherry mRNA control N/A 4.1 95.9 MOC transfection control N/A 3.0 97.0

The following data were obtained when using the excision strategy withdual sgRNAs:

When IVS40 USH2A mutant cells were transfected with a first syntheticsgRNA, USH2Amut_T176 (a sgRNA comprising SEQ ID NO: 5290), and a secondsynthetic sgRNA, USH2Amut_T715 (a sgRNA comprising SEQ ID NO: 5300),77.5% of USH2A transcripts were free of the aberrant intronic sequence(pseudo-exon) caused by the IVS40 mutation.

When IVS40 USH2A mutant cells were transfected with a first syntheticsgRNA, USH2Amut_T210 (a sgRNA comprising SEQ ID NO: 5294), and a secondsynthetic sgRNA, USH2Amut_T715 (a sgRNA comprising SEQ ID NO: 5300),67.3% of USH2A transcripts were free of the aberrant intronic sequence(pseudo-exon) caused by the IVS40 mutation.

When IVS40 USH2A mutant cells were transfected with a first syntheticsgRNA, USH2Amut_T387 (a sgRNA comprising SEQ ID NO: 5295), and a secondsynthetic sgRNA, USH2Amut_T715 (a sgRNA comprising SEQ ID NO: 5300),77.6% of USH2A transcripts were free of the aberrant intronic sequence(pseudo-exon) caused by the IVS40 mutation.

When IVS40 USH2A mutant cells were transfected with a first syntheticsgRNA, USH2Amut_T193 (a sgRNA comprising SEQ ID NO: 5277), and a secondsynthetic sgRNA, USH2Amut_T343 (a sgRNA comprising SEQ ID NO: 5279),62.6% of USH2A transcripts were free of the aberrant intronic sequence(pseudo-exon) caused by the IVS40 mutation.

When IVS40 USH2A mutant cells were transfected with a first syntheticsgRNA, USH2Amut_T387 (a sgRNA comprising SEQ ID NO: 5295), and a secondsynthetic sgRNA, USH2Amut_T343 (a sgRNA comprising SEQ ID NO: 5279),77.4% of USH2A transcripts were free of the aberrant intronic sequence(pseudo-exon) caused by the IVS40 mutation.

When IVS40 USH2A mutant cells were transfected with a first syntheticsgRNA, USH2Amut_T505 (a sgRNA comprising SEQ ID NO: 5274), and a secondsynthetic sgRNA, USH2Amut_T343 (a sgRNA comprising SEQ ID NO: 5279),48.5% of USH2A transcripts were free of the aberrant intronic sequence(pseudo-exon) caused by the IVS40 mutation.

When IVS40 USH2A mutant cells were transfected with a first syntheticsgRNA, USH2Amut_T585 (a sgRNA comprising SEQ ID NO: 5275), and a secondsynthetic sgRNA, USH2Amut_T343 (a sgRNA comprising SEQ ID NO: 5279),71.9% of USH2A transcripts were free of the aberrant intronic sequence(pseudo-exon) caused by the IVS40 mutation.

When IVS40 USH2A mutant cells were mock transfected with no RNP complex,1.0% of USH2A transcripts were free of the aberrant intronic sequence(pseudo-exon) caused by the IVS40 mutation.

The following data were obtained when using the NHEJ strategy withsingle sgRNAs:

When IVS40 USH2A mutant cells were transfected with a synthetic sgRNA,USH2A MP (a sgRNA comprising SEQ ID NO: 5327), 90.7% of USH2Atranscripts were free of the aberrant intronic sequence (pseudo-exon)caused by the IVS40 mutation.

When IVS40 USH2A mutant cells were transfected with a synthetic sgRNA,USH2A MO (a sgRNA comprising SEQ ID NO: 5321), 89.8% of USH2Atranscripts were free of the aberrant intronic sequence (pseudo-exon)caused by the IVS40 mutation.

When IVS40 USH2A mutant cells were transfected with a synthetic sgRNA,USH2A MG (a sgRNA comprising SEQ ID NO: 5323), 37.4% of USH2Atranscripts were free of the aberrant intronic sequence (pseudo-exon)caused by the IVS40 mutation.

When IVS40 USH2A mutant cells were transfected with a synthetic sgRNA,USH2A MB (a sgRNA comprising SEQ ID NO: 5325), 43.3% of USH2Atranscripts were free of the aberrant intronic sequence (pseudo-exon)caused by the IVS40 mutation.

When IVS40 USH2A mutant cells were transfected with a synthetic sgRNA,USH2A MR (a sgRNA comprising SEQ ID NO: 5328), 84.1% of USH2Atranscripts were free of the aberrant intronic sequence (pseudo-exon)caused by the IVS40 mutation.

When IVS40 USH2A mutant cells were transfected with a GFP plasmidcontrol, 1.9% of USH2A transcripts were free of the aberrant intronicsequence (pseudo-exon) caused by the IVS40 mutation.

When IVS40 USH2A mutant cells were transfected with a mCherry mRNAcontrol, 4.1% of USH2A transcripts were free of the aberrant intronicsequence (pseudo-exon) caused by the IVS40 mutation.

When IVS40 USH2A mutant cells were transfected with an MOC transfectioncontrol, 3.0% of USH2A transcripts were free of the aberrant intronicsequence (pseudo-exon) caused by the IVS40 mutation.

These data provide evidence that splicing of transcripts from a USH2Agene comprising an IVS40 mutation can be corrected via either the NHEJstrategy provided herein or the excision strategy provided herein,leading to expression of functional USH2A protein.

Example 23 Testing of Guide RNAs in Cells for On-Target and Off-TargetActivity

To further evaluate gRNAs provided herein, selected gRNAs could befurther tested for on-target activity in immortalized humanpatient-derived fibroblasts that have homozygous IVS40 mutations of theUSH2A gene.

Patients having homozygous IVS40 mutations in an allele of the USH2Agene provide skin biopsies to create an immortalized cell line. Theimmortalized cell line is transfected with a sgRNA and a Cas9 (ornucleic acid encoding the sgRNA and/or Cas9). Both the NHEJ and theexcision strategies are tested. Analysis of targeted genome deletionand/or splicing of the USH2A transcripts in edited cells is performed byddPCR as described herein. Results could indicate the ability of genomeediting according to the present disclosure to correct the IVS40mutation leading to expression of functional USH2A protein inpatient-derived cells.

Similar tests are conducted on immortalized cells from healthy humanvolunteers to analyze off-target activity for gRNAs of the presentdisclosure.

Example 24 Testing of Other gRNAs

Additional sgRNAs comprising other spacer sequences such as, forexample, AUAUGAUGAUAGUAUUAU (SEQ ID NO: 5443), are tested usingexperiments such as those corresponding to those in Examples describedherein. SEQ ID NO: 5443 is a spacer sequence found in FIG. 2G, thetarget DNA sequence (5′-3′) is found in FIG. 2H, and the reverse strandof the target DNA sequence to which the sgRNA will bind (5′-3′) is foundin FIG. 2I.

For example, on-target and off-target activity is determined using celllines and methods described herein. The ability of such an sgRNA tocorrect the USH2A IVS40 splicing defect is tested using the BFP splicingreporter assay (exemplified in Examples 18-21) and/or a ddPCR assay(exemplified in Example 22). Additional tests are performed inpatient-derived cells (Example 23) to confirm the ability of suchsequences to be used for editing. sgRNAs comprising other spacersequences could be paired with either each other or with other sgRNAsprovided herein and used as dual sgRNAs. sgRNAs comprising other spacersequences could be used as single sgRNAs.

Example 25 Testing Different Approaches for HDR Gene Editing

In addition to testing a gRNA for on-target activity and off-targetactivity, the HDR strategy is tested.

For the HDR strategy, donor DNA template are provided as a shortsingle-stranded oligonucleotide, a short double-stranded oligonucleotide(PAM sequence intact/PAM sequence mutated), a long single-stranded DNAmolecule (PAM sequence intact/PAM sequence mutated) or a longdouble-stranded DNA molecule (PAM sequence intact/PAM sequence mutated).In some examples, the donor DNA template is delivered by AAV.

These results demonstrate the efficacy of the various HDR gene editingstrategies and are used to select effective constructs and strategies.

Example 26 Re-Assessment of Lead CRISPR-Cas9/DNA Donor Combinations

In some cases, one or more CRISPR-Cas9/DNA donor combinations arere-assessed in cells for efficiency of deletion, recombination, andoff-target specificity. In some embodiments, Cas9 mRNA or RNP areformulated into lipid nanoparticles for delivery, sgRNAs are formulatedinto nanoparticles or delivered as a recombinant AAV particle, and donorDNA are formulated into nanoparticles or delivered as recombinant AAVparticle.

These data demonstrate the efficacy of a formulation for, e.g., an HDRgene editing strategy.

Example 27 In Vivo Testing in Relevant Animal Model

Selected CRISPR-Cas9/gRNA combinations identified herein are tested invivo in an animal model, such as a rhesus macaque (Macaca mulatta) andcrab-eating macaque (Macaca fascicularis). In addition, formulations aretested in a human eye from a human donor.

NOTE REGARDING ILLUSTRATIVE EXAMPLES

While the present disclosure provides descriptions of various specificaspects for the purpose of illustrating various examples of the presentdisclosure and/or its potential applications, it is understood thatvariations and modifications will occur to those skilled in the art.Accordingly, the invention or inventions described herein should beunderstood to be at least as broad as they are claimed, and not as morenarrowly defined by particular illustrative examples provided herein.

1.-137. (canceled)
 138. A method for editing a USH2A gene comprising anIVS40 mutation, comprising: introducing into a cell: (i) one or more S.pyogenes Cas9 endonucleases and one or more guide RNAs (gRNAs)comprising a spacer sequence selected from the group consisting ofnucleic acid sequences in SEQ ID NOs: 5272-5319, 5321, 5323, 5325,5327-5328 and 5443; or (ii) one or more S. aureus Cas9 endonucleases andone or more guide RNAs (gRNAs) comprising a spacer sequence selectedfrom the group consisting of nucleic acid sequences in SEQ ID NOs:5446-5461, thereby effecting one or more single stranded breaks (SSBs)or double stranded breaks (DSBs) within or near intron 40 of the USH2Agene that results in an edited human cell.
 139. The method of claim 138,wherein the IVS40 mutation is located within intron 40 of the USH2Agene.
 140. The method of claim 138, wherein the method comprisesintroducing into the cell one or more polynucleotides encoding the oneor more endonucleases.
 141. The method of claim 138, wherein the one ormore gRNAs are single-molecule guide RNAs (sgRNAs).
 142. The method ofclaim 138, wherein the one or more DNA endonucleases is pre-complexedwith one or more gRNAs.
 143. The method of claim 141, wherein the one ormore DNA endonucleases is pre-complexed with one or more sgRNAs.
 144. Amethod for editing a USH2A gene comprising an IVS40 mutation,comprising: introducing into a cell: (i) one or more S. pyogenes Cas9endonucleases and a first gRNA and a second gRNA selected from the groupconsisting of: (a) a first gRNA comprising SEQ ID NO: 5321 and a secondgRNA comprising any one of SEQ ID NOs: 5267-5269; (b) a first gRNAcomprising SEQ ID NO: 5323 and a second gRNA comprising any one of SEQID NOs: 5267-5269; (c) a first gRNA comprising SEQ ID NO: 5325 and asecond gRNA comprising any one of SEQ ID NOs: 5267-5269; (d) a firstgRNA comprising SEQ ID NO: 5327 and a second gRNA comprising any one ofSEQ ID NOs: 5267-5269; (e) a first gRNA comprising SEQ ID NO: 5329 and asecond gRNA comprising any one of SEQ ID NOs: 5267-5269; (f) a firstgRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO:5279; (g) a first gRNA comprising SEQ ID NO: 5294 and a second gRNAcomprising SEQ ID NO: 5300; (h) a first gRNA comprising SEQ ID NO: 5295and a second gRNA comprising SEQ ID NO: 5300; (i) a first gRNAcomprising SEQ ID NO: 5290 and a second gRNA comprising SEQ ID NO: 5300;and (j) a first gRNA comprising SEQ ID NO: 5277 and a second gRNAcomprising SEQ ID NO: 5300, or (ii) one or more S. aureus Cas9endonucleases and a first gRNA and a second gRNA selected from the groupconsisting of: (a) a first gRNA comprising SEQ ID NO: 5452 and a secondgRNA comprising SEQ ID NO: 5449; (b) a first gRNA comprising SEQ ID NO:5453 and a second gRNA comprising SEQ ID NO: 5449; (c) a first gRNAcomprising SEQ ID NO: 5455 and a second gRNA comprising SEQ ID NO: 5457;(d) a first gRNA comprising SEQ ID NO: 5452 and a second gRNA comprisingSEQ ID NO: 5451; and (e) a first gRNA comprising SEQ ID NO: 5448 and asecond gRNA comprising SEQ ID NO: 5449, thereby effecting one or moresingle stranded breaks (SSBs) or double stranded breaks (DSBs) within ornear intron 40 of the USH2A gene that results in an edited human cell.145. The method of claim 144, wherein the IVS40 mutation is locatedwithin intron 40 of the USH2A gene.
 146. The method of claim 144,wherein the method comprises introducing into the cell one or morepolynucleotides encoding the one or more endonucleases.
 147. The methodof claim 144, wherein the gRNAs are sgRNAs.
 148. The method of claim144, wherein the one or more DNA endonucleases is pre-complexed with oneor more gRNAs.
 149. The method of claim 147, wherein the one or more DNAendonucleases is pre-complexed with one or more sgRNAs.
 150. A gRNAcomprising a spacer sequence selected from the group consisting ofnucleic acid sequences in SEQ ID NOs: 5272-5319, 5321, 5323, 5325,5327-5328, 5443, and 5446-5461.
 151. The gRNA of claim 150, wherein thegRNA is a sgRNA.
 152. A method for treating a patient with UsherSyndrome Type 2A, comprising administering to the patient (i) one ormore endonucleases selected from a S. pyogenes Cas9 endonuclease and aS. aureus Cas9 endonuclease and (ii) one or more gRNAs of claim 150,wherein the patient comprises an IVS40 mutation in an USH2A gene.
 153. Amethod for treating a patient with Usher Syndrome Type 2A, comprisingadministering to the patient a cell edited ex vivo by a methodcomprising introducing into the cell (i) one or more endonucleasesselected from a S. pyogenes Cas9 endonuclease and a S. aureus Cas9endonuclease and (ii) one or more gRNAs of claim 150, thereby effectingone or more single stranded breaks (SSBs) or double stranded breaks(DSBs) within or near intron 40 of an USH2A gene that results in anedited cell, wherein the patient comprises an IVS40 mutation in theUSH2A gene.
 154. A composition comprising at least two gRNAs selectedfrom the group consisting of: (a) a first gRNA comprising SEQ ID NO:5321 and a second gRNA comprising any one of SEQ ID NOs: 5267-5269; (b)a first gRNA comprising SEQ ID NO: 5323 and a second gRNA comprising anyone of SEQ ID NOs: 5267-5269; (c) a first gRNA comprising SEQ ID NO:5325 and a second gRNA comprising any one of SEQ ID NOs: 5267-5269; (d)a first gRNA comprising SEQ ID NO: 5327 and a second gRNA comprising anyone of SEQ ID NOs: 5267-5269; (e) a first gRNA comprising SEQ ID NO:5329 and a second gRNA comprising any one of SEQ ID NOs: 5267-5269; (f)a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQID NO: 5279; (g) a first gRNA comprising SEQ ID NO: 5294 and a secondgRNA comprising SEQ ID NO: 5300; (h) a first gRNA comprising SEQ ID NO:5295 and a second gRNA comprising SEQ ID NO: 5300; (i) a first gRNAcomprising SEQ ID NO: 5290 and a second gRNA comprising SEQ ID NO: 5300;(j) a first gRNA comprising SEQ ID NO: 5277 and a second gRNA comprisingSEQ ID NO: 5300; (k) a first gRNA comprising SEQ ID NO: 5452 and asecond gRNA comprising SEQ ID NO: 5449; (1) a first gRNA comprising SEQID NO: 5453 and a second gRNA comprising SEQ ID NO: 5449; (m) a firstgRNA comprising SEQ ID NO: 5455 and a second gRNA comprising SEQ ID NO:5457; (n) a first gRNA comprising SEQ ID NO: 5452 and a second gRNAcomprising SEQ ID NO: 5451; and (o) a first gRNA comprising SEQ ID NO:5448 and a second gRNA comprising SEQ ID NO:
 5449. 155. The compositionof claim 154, wherein the gRNAs are sgRNAs.
 156. A method for treating apatient with Usher Syndrome Type 2A, comprising administering to thepatient (i) one or more endonucleases selected from a S. pyogenes Cas9endonuclease and a S. aureus Cas9 endonuclease and (ii) the compositionof claim 154, wherein the patient comprises an IVS40 mutation in anUSH2A gene.
 157. A method for treating a patient with Usher SyndromeType 2A, comprising administering to the patient a cell edited ex vivoby a method comprising introducing into the cell (i) one or moreendonucleases selected from a S. pyogenes Cas9 endonuclease and a S.aureus Cas9 endonuclease and (ii) the composition of claim 154, therebyeffecting one or more single stranded breaks (SSBs) or double strandedbreaks (DSBs) within or near intron 40 of an USH2A gene that results inan edited cell, wherein the patient comprises an IVS40 mutation in theUSH2A gene.